Heather Medbury

The role of the fibrocyte in intimal hyperplasia

Summary.  Background: Experimental animal studies have shown that the intimal hyperplasia (IH) responsible for occlusion after successful revascularization procedures may be partially caused by a bone marrow‐derived cell that migrates to the site of vascular injury. Concurrent studies have demonstrated an extensive role in wound healing for the circulating fibrocyte. Objectives: We aimed to trace the path of the circulating cell that contributes to IH and determine if it is the fibrocyte. Methods and results: We established an in vitro model whereby purified monocytes from six healthy human volunteers were cultured into fibrocytes. These cells were morphometrically similar to the vascular smooth muscle cell (VSMC) found in IH and expressed alpha‐smooth muscle actin (α‐SMA) as well as CD34, CD45 and Collagen I (Col I), markers indicative of the fibrocyte. In an in vivo ovine carotid artery synthetic patch graft model, carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeled circulating leukocytes were observed throughout the graft as well as in the neointima in 18 sheep. These cells were shown to produce collagen and α‐SMA at 1, 2 and 4 weeks. These cells then underwent immunohistochemical analysis and were found to express a set of markers unique to the fibrocyte (CD34, CD45, Vimentin and α‐SMA) and also to double stain for CD34 and α‐SMA. Conclusions: IH in an ovine carotid artery patch graft model is partially derived from a hematopoietic circulating progenitor cell that acquires mesenchymal features as it matures at the site of injury.

The cytokine and histological response in islet xenograft rejection is dependent upon species combination.

BACKGROUND Islet xenografts have clinical potential, may avoid hyperacute rejection, and therefore are a good place to examine the cellular xenograft immune response. The aim of this study was to examine the cellular, humoral, and cytokine response in islet xenograft rejection and to determine the difference in the immune response with a different donor species. METHODS Two islet xenograft models (DA rat islets to B6AF1 mouse and canine islets to B6AF1 mouse) and a mouse syngeneic control model were examined histologically and by a semiquantitative polymerase chain reaction method. RESULTS There was significant up-regulation of all intragraft cytokines tested (interleukin [IL]-2, IL-4, IL-5, IL-10, and interferon-gamma) in both xenograft models compared with the controls. However, the dog islet grafts had higher levels of IL-4 and IL-5 gene expression than the rat islet grafts, which, conversely, had higher levels of interferon-gamma gene expression. These differences correlated with the histological and anti-donor antibody production differences between the two models. The dog to mouse model had an intense eosinophilic infiltrate and an early up-regulation of anti-donor antibody, whereas there was little eosinophilic infiltrate and a delayed anti-donor antibody up-regulation in the rat to mouse model. CONCLUSIONS The mouse used different mechanisms to reject the rat and canine islets, suggesting that the immune response in islet xenograft rejection may be dependent on the species combination. It may not be possible to characterize the cellular xenograft rejection response in a bipolar manner as has been the case with humoral rejection response. Caution therefore needs to be taken before extrapolating the cellular immune responses seen in animal models to the clinical setting.

Calcium channel antagonist verapamil inhibits neointimal formation and enhances apoptosis in a vascular graft model

BACKGROUND The potential of the calcium channel antagonist verapamil to cause apoptosis (programmed cell death) is of considerable importance in arterial injury where the loss of smooth muscle cells may contribute to a reduction in intimal hyperplasia development. The aim of this study was to determine whether verapamil induces vascular cell apoptosis after carotid artery synthetic grafting. METHODS Thirty-two adult-female Merino sheep received gelatin sealed fusiform shape-Dacron grafts into the left common carotid artery at day 0. After operation animals were randomly allocated to either a control group or one of three treatment groups (groups 2, 3, and 4). Group 1 animals (n = 9) received no treatment. For the treatment groups, intravenous verapamil was given at a rate of 0.5 mg/kg per day in two divided doses. Group 2, 3, and 4 sheep were treated for 1, 2, and 4 weeks, respectively. Animals were sacrificed at 4 weeks. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-fluorescent labelling. Proliferating cells and their phenotype were determined by doublestaining with antiproliferation cellular nuclei antigen and anti-alpha-actin or anti-HAM-56. RESULTS There were significantly more apoptotic cells in the perigraft adventitia in the 4-week treatment group than in the control group (P <0.05). The average number of proliferating cells at 2 and 4 weeks in the intima were significantly less than in the control (P <0.05). The average numbers of macrophages inside graft matrix in the 2 and 4 weeks treatment groups were significantly less than for the control (P <0.05). The number of proliferating cells inside the graft was significantly lower at 4 weeks compared with control (P <0.05). There was negative correlation between intimal PCNA expression and perigraft apoptotic expression level (P <0.05). CONCLUSION The antihypertensive agent verapamil inhibits intimal hyperplasia through enhancing adventitial cell apoptosis and inhibiting intimal cell proliferation after vascular grafting.

Induction of contact-dependent endothelial apoptosis by osteosarcoma cells suggests a role for endothelial cell apoptosis in blood-borne metastasis.

Although tumour cells are believed to migrate between endothelial cells early in metastasis, the possibility remains that endothelial apoptosis may also contribute to the tumours breach of the vascular barrier. Although seemingly inconsistent with tumour angiogenesis, one publication describes the induction of contact‐dependent apoptosis in cultured endothelium by tumour cells. The cell culture data are, however, open to challenge on technical grounds while there are no confirmatory reports. The present paper describes experiments overcoming these limitations. SAOS‐2 human osteosarcoma cells and two rat carcinoma cell lines were co‐cultured with human umbilical vein endothelial cells (HUVECs) and cultures labelled by surface lectin histochemistry for endothelium. The HUVEC culture density was determined and SAOS‐2 cells, but not rat carcinoma cells, were found significantly to reduce HUVEC survival despite the release of potent growth factors as determined in separate experiments with tumour cell conditioned medium. Lectin labelling combined with light microscopy, transmission electron microscopy, flow cytometry for both lectin binding and DNA content, and DNA gel electrophoresis of SAOS‐2/HUVEC co‐cultures revealed extensive HUVEC apoptosis. These findings indicate contact‐dependent endothelial apoptosis by SAOS‐2, while this activity appeared weaker and overwhelmed by HUVEC proliferation with rat carcinoma cells. Importantly, this study supports the suggestion that endothelial apoptosis may be important for metastasis and suggests a complex interplay between endothelial proliferation and apoptosis in tumours. Copyright

Clinical significance of macrophage phenotypes in cardiovascular disease

The emerging understanding of macrophage subsets and their functions in the atherosclerotic plaque has led to the consensus that M1 macrophages are pro-atherogenic while M2 macrophages may promote plaque stability, primarily though their tissue repair and anti-inflammatory properties. As such, modulating macrophage function to promote plaque stability is an exciting therapeutic prospect. This review will outline the involvement of the different macrophage subsets throughout atherosclerosis progression and in models of regression. It is evident that much of our understanding of macrophage function comes from in vitro or small animal models and, while such knowledge is valuable, we have much to learn about the roles of the macrophage subsets in the clinical setting in order to identify the key pathways to target to possibly promote plaque stability.

Lack of increased expression of cell surface markers for circulating fibrocyte progenitors in limited scleroderma.

The aetiology and pathogenesis of scleroderma is incompletely understood. Recently, a cell called the fibrocyte has been shown to be derived from circulating monocytes with the ability to produce collagen. The aim of this study was to evaluate differences in the cell surface characteristics of circulating fibrocyte progenitors (monocytes) in patients with limited scleroderma compared to controls. A case-control study was performed in eight patients with limited scleroderma, which were matched with eight controls. Three-colour flow cytometry was used to assess the relative expression of cell surface markers. Statistical analysis then compared the relative expression between the two groups. In this preliminary study, there were no significant differences in the expression of circulating monocyte surface molecules involved with cell transformation, function, or migration presumed to give rise to fibrocytes, in a population of patients with limited scleroderma. Various explanations for the results are discussed.

Are fibrocytes present in pediatric burn wounds

Objective of the study is to investigate for the presence of fibrocytes, a leucocyte found at sites of injury with fibroblast-like properties, within pediatric burn wounds. Seventy 3 mm punch biopsies were taken from 53 burn wounds in 33 children between 7 months and 15 years of age at the time of planned operative debridement and grafting. After fixation and sectioning, immunohistochemistry (IHC) staining was performed for CD34, pro-collagen I, &agr; smooth muscle actin, transforming growth factor &bgr;1 and Leucocyte Specific Protein-1 (LSP-1). The presence of fibrocytes was confirmed by double immunofluorescence staining with antibodies to CD34 or LSP-1 with pro-collagen I. CD34 positive cells were present in all burn wound biopsies. Using IHC staining, in 18 patients cells positive for CD34 and pro-collagen I were identified; in 17 patients, cells positive for CD34 and &agr;SMA and in 17 patients also cells positive for LSP-1 and pro-collagen I. Double immunofluorescence for CD34/pro-collagen I and LSP-1/pro-collagen I confirmed the presence of fibrocytes in specimens from 17 of 18 patients positive for these markers on IHC. Of the 17 patients whose burn wounds were complicated by hypertrophic scarring, fibrocytes were identified in 88% (n = 15) compared with 18% of those without hypertrophic scarring (P < .001). This study represents the first report of the presence of fibrocytes in acute pediatric burn wounds. These cells appear to be involved in the local response to burn wound injury and may correlate with the later development of hypertrophic burn wound scarring.

The anti-apoptotic activity of albumin for endothelium is inhibited by advanced glycation end products restricting intramolecular movement

Human serum albumin (HSA) inhibits endothelial apoptosis in a highly specific manner. CNBr fragmentation greatly increases the effectiveness of this activity, suggesting that this type of protection is mediated by a partially cryptic albumin domain which is transiently exposed by intramolecular movement. Advanced glycation end-product (AGE) formation in HSA greatly reduces its intra-molecular movement. This study aimed to determine if this inhibits the anti-apoptotic activity of HSA, and if such inactivation could be reversed by CNBr fragmentation. HSA-AGE was prepared by incubating HSA with glucose, and assessed using the fructosamine assay, mass spectrometry, SDS-PAGE and fluorometry. Low levels of AGE in the HSA had little effect upon its anti-apoptotic activity, but when the levels of AGE were high and the intra-molecular movement was reduced, endothelial cell survival was also found to be reduced to levels equivalent to those in cultures without HSA or serum (p > 0.001). Survival was restored by the inclusion of native HSA, despite the presence of HSA with high levels of AGE. Also, CNBr fragmentation of otherwise inactive HSA-AGE restored the anti-apoptotic activity for endothelium. Apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and fluorescence-activated cell sorting analysis, and there was no evidence for direct toxicity in the HSA-AGE preparations. The results are consistent with the proposed role of intra-molecular movement in exposing the anti-apoptotic domain in HSA for endothelium. The levels of AGE formation required to inhibit the anti-apoptotic activity of HSA exceeded those reported for diabetes. Nonetheless, the data from this study seems to be the first example of reduced protein function due to AGE-restricted intra-molecular movement.

The Bidirectional Relationship between Cholesterol and Macrophage Polarization

Whether an atherosclerotic plaque progresses and eventually ruptures is heavily influenced by the function of macrophages. However, it is clear that a spectrum of macrophage phenotypes is present in the plaque, with some exhibiting stabilizing functions. While macrophages expressing characteristic M1 and M2 markers are evident, the disparate microenvironments of the plaque, such as regions of hemorrhage, promote other distinct macrophage phenotypes. Crucial to plaque development and progression is macrophage exposure to accumulated modified low density lipoproteins that leads to foam cell formation and development of the necrotic core. There are a range of biologically active compounds in low density lipoprotein (LDL) each having some bearing on which macrophage surface receptors are engaged and what cellular response ensues. Understanding the bidirectional interplay between ‘cholesterol’ and macrophage phenotype will provide valuable insight into key pathways to target which may possibly promote plaque stability by modulating macrophage function.

CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis

Increasing evidence shows that CC‐chemokines promote inflammatory‐driven angiogenesis, with little to no effect on hypoxia‐mediated angiogenesis. Inhibition of the CC‐chemokine class may therefore affect angiogenesis differently depending on the pathophysiological context. We compared the effect of CC‐chemokine inhibition in inflammatory and physiological conditions. In vitro, the broad‐spectrum CC‐chemokine inhibitor “35K” inhibited inflammatory‐induced endothelial cell proliferation, migration, and tubulogenesis, with more modest effects in hypoxia. In vivo, adenoviruses were used to overexpress 35K (Ad35K) and GFP (AdGFP, control virus). Plasma chemokine activity was suppressed by Ad35K in both models. In the periarterial femoral cuff model of inflammatory‐driven angiogenesis, overexpression of 35K inhibited adventitial neovessel formation compared with control AdGFP‐infused mice. In contrast, 35K preserved neovascularization in the hindlimb ischemia model and had no effect on physiological neovascularization in the chick chorioallantoic membrane assay. Mechanistically, 2 key angiogenic proteins (VEGF and hypoxia‐inducible factor‐1α) were conditionally regulated by 35K, such that expression was inhibited in inflammation but was unchanged in hypoxia. In conclusion, CC‐chemokine inhibition by 35K suppresses inflammatory‐driven angiogenesis while preserving physiological ischemia‐mediated angiogenesis via conditional regulation of VEGF and hypoxia‐inducible factor‐1α. CC‐chemokine inhibition may be an alternative therapeutic strategy for suppressing diseases associated with inflammatory angiogenesis without inducing the side effects caused by global inhibition.—Ridiandries, A., Tan, J. T. M., Ravindran, D., Williams, H., Medbury, H. J., Lindsay, L., Hawkins, C., Prosser, H. C. G., Bursill, C. A. CC‐chemokineclass inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis. FASEB J. 31, 1179–1192 (2017). www.fasebj.org