Monocyte M1/M2 profile is altered in paediatric burn patients with hypertrophic scarring.

Hypertrophic scars (HTS) remain a common outcome of burn injury, particularly in children. They can arise from variations in the wound healing stages, such as an excessive inflammatory response or inefficient remodelling. Of the cells contributing to these healing stages, macrophages and fibrocytes are crucial. Specifically, the inflammatory phase is dominated by M1 macrophages, the proliferation/remodelling stages by M2 macrophages, and scar tissue contains numerous fibrocytes. As the progenitors to these cells, monocytes, can also exhibit M1- and M2-skewing, we proposed that their profile, or circulating fibrocyte counts, could be used to predict poor healing outcomes. To investigate this, we obtained blood samples from paediatric controls and burns patients, which were then divided into HTS and NoHTS groups upon scar assessment at 12 months. The samples were assessed by whole blood flow cytometry to quantify fibrocytes and monocyte subset proportions and to determine monocyte levels of M1 (CD86, CD120b, CD319) and M2 (CD93, CD163, CD200R) markers. Both burns groups had higher proportions of classical monocytes compared to controls, indicating increased cell turnover and/or entry of other subsets into the wound. In burns patients who took more than 21 days to heal, the HTS group had lower M2 (CD200R) expression with the ratio of M1/M2 (CD86/CD200R) being significantly higher. These results suggest an elevated early inflammatory monocyte response contributes to development of HTS. Correlations of marker expression with remaining healing time revealed a significant positive correlation with M1 (CD120b) and M1/M2 (CD120b/CD200R), suggesting a potential role for CD120b as an indicator of healing delay. Fibrocytes did not significantly differ between the groups. In conclusion, increased monocyte inflammation likely contributes to slower healing and development of scarring, but further studies are needed to determine the predictive power of monocyte inflammatory profile.

Macrophages bind LDL using heparan sulfate and the perlecan protein core.

The retention of low-density lipoprotein (LDL) is a key process in the pathogenesis of atherosclerosis and largely mediated via smooth-muscle cell-derived extracellular proteoglycans including the glycosaminoglycan chains. Macrophages can also internalize lipids via complexes with proteoglycans. However, the role of polarized macrophage-derived proteoglycans in binding LDL is unknown and important to advance our understanding of the pathogenesis of atherosclerosis. We therefore examined the identity of proteoglycans, including the pendent glycosaminoglycans, produced by polarized macrophages to gain insight into the molecular basis for LDL binding. Using the quartz crystal microbalance with dissipation monitoring technique, we established that classically activated macrophage (M1)- and alternatively activated macrophage (M2)-derived proteoglycans bind LDL via both the protein core and heparan sulfate (HS) in vitro. Among the proteoglycans secreted by macrophages, we found perlecan was the major protein core that bound LDL. In addition, we identified perlecan in the necrotic core as well as the fibrous cap of advanced human atherosclerotic lesions in the same regions as HS and colocalized with M2 macrophages, suggesting a functional role in lipid retention in vivo. These findings suggest that macrophages may contribute to LDL retention in the plaque by the production of proteoglycans; however, their contribution likely depends on both their phenotype within the plaque and the presence of enzymes, such as heparanase, that alter the secreted protein structure.

Monocyte inflammatory profile is specific for individuals and associated with altered blood lipid levels.

BACKGROUND AND AIMS: Atherogenesis is dependent upon monocyte influx into the vessel wall. In humans, three monocyte subsets exist, the number and function of which are significantly altered in cardiovascular disease (CVD). Whether such alterations arise in individuals with a perturbed lipid profile remains largely unanswered, but is important to delineate, as adoption of a pro-inflammatory state may promote plaque formation. Here, we compared the inflammatory status of monocyte subsets and determined whether monocyte inflammatory changes are evident in individuals with a perturbed lipid profile. METHODS: Monocyte subset cytokine production, inflammatory and anti-inflammatory marker expression were determined by whole blood flow cytometry and related to participants' lipid levels. RESULTS: The intermediate and non-classical monocytes were more inflammatory than classicals as seen by their higher cytokine production (TNF-α, IL-1ß, IL-6) and M1 marker (CD86) expression, but lower levels of M2 markers (CD93, CD163). More importantly, a considerable variation was seen between participants, with all monocytes of one individual being more inflammatory than those of another. Many inter-individual differences were related to participants' lipid levels. IL-1ß production correlated negatively with Apo A1 and HDL-C. CD86 and TLR2 correlated positively with Chol:HDL-C but negatively with HDL-C and Apo A1:Apo B. Interestingly, CD163 expression correlated positively with Chol:HDL-C but negatively with Apo A1:Apo B. CONCLUSIONS: Our data indicates that priming of all monocytes to an inflammatory state occurs in individuals with a perturbed lipid profile, overriding the normal functional distinction attributed to the different monocyte subsets. As such, all monocytes may be important in CVD.

Human classical monocytes display unbalanced M1/M2 phenotype with increased atherosclerotic risk and presence of disease.

BACKGROUND: Specific monocyte and macrophage subsets have been implicated in atherosclerosis, with intermediate monocytes proportionally elevated in cardiovascular disease and M1 macrophages abundant in unstable atherosclerotic plaques. While several studies have shown altered proportions of these subsets in atherosclerosis, studies examining functional and phenotypic subset alterations remain scarce. METHODS: We used whole blood flow cytometry to investigate the expression of M1 (CD86) and M2 (CD163) markers on monocyte subsets of atherosclerotic patients and controls. RESULTS: Atherosclerotic patients had a more inflammatory monocyte profile than controls, indicated by increased intermediate subset proportions, a higher classical monocyte CD86/CD163 ratio, and elevated serum M1-related chemokines. A more inflammatory profile appeared to correlate with atherosclerotic risk, as in controls classical monocyte CD86/CD163 ratio was negatively correlated with HDL and apolipoprotein A1, and positively correlated with interleukin-1ß. CONCLUSIONS: We conclude that monocyte subsets show functional and phenotypic changes in cardiovascular disease and such changes are likely to contribute to atherosclerotic progression.

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

A Review of Monocytes and Monocyte-Derived Cells in Hypertrophic Scarring Post Burn.

Pediatric burns remain a common injury after which many patients develop a severe form of scarring known as hypertrophic scarring. The formation of the hypertrophic scar arises from excessive production of collagen during wound healing. Wound repair and regeneration represents a complex process that is accomplished through many biological processes involving various cell types, extracellular matrix proteins, cytokines, and other mediators. One important cell type is the monocyte, which displays an altered profile in many wound models. These profile changes may function as biomarkers, reflecting and/or influencing the clinical outcome of the healing response seen after burn injury. Monocytes circulate in the blood and then enter into the tissue, where they further differentiate into macrophages, which serve various functions, including immune defense and tissue remodeling. More recently, these cells have been characterized in detail based on cell surface markers expressed and genes up-regulated, enabling subpopulations to be identified. Fibrocytes, which are also monocyte-derived cells, have been shown to contribute to collagen production in the burn wound and are associated with hypertrophic scarring. They may represent a unique subpopulation of macrophages that, due to their production of collagen, promote tissue fibrosis rather than wound repair. A better understanding of the relationship among monocytes, fibrocytes, and macrophages may improve our appreciation of the factors influencing scar formation and tissue remodeling.

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, alpha smooth muscle actin, transforming growth factor beta1 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 alphaSMA 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.

Perigraft adventitia and intima remodeling after synthetic patch implantation in sheep carotid artery: role of apoptosis and proliferation.

BACKGROUND: The mechanisms of neointima formation after synthetic vascular grafting are not clear. The aim of this study was to investigate the intima and perigraft adventitia remodeling process in terms of cell apoptosis versus proliferation after synthetic patch implantation. METHODS: Female Merino sheep were randomized equally into two groups and underwent implantion with a patch of gelatin sealed Dacron graft into the left common carotid artery. At 1 and 6 months, grafted vessels were harvested, processed, and assessed. Intimal area and lumen sizes were measured with histologic assessment of eight segments from each animal assisted with image analysis. Immunohistochemical labeling of alpha-actin and D33 desmin was performed on tissue sections of perigraft adventitia, graft matrix, and intima. Cell proliferation and cell phenotype were determined with double immunohistochemical staining with anti-proliferating cell nuclear antigen and anti-alpha-actin or antimacrophage antibodies (HAM 56) in perigraft adventitia, graft matrix, and intima. Apoptosis was detected with in situ terminal deoxynucleiotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-fluorescence nick end labeling (TUNEL) in perigraft adventitia, graft matrix, and intima. RESULTS: The carotid artery lumen size at 6 months was significantly larger than at 1 month (P

Synergistic induction of apoptosis in human endothelial cells by tumour necrosis factor-alpha and transforming growth factor-beta.

Serum deprivation stimulates endothelial apoptosis while albumin inhibits this and has been proposed as important in confining apoptotic remodelling to poorly perfused vessels. Tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta are also reported to induce endothelial apoptosis. To investigate the comparative roles of these stimuli, the effect of TNF-alpha and TGF-beta, alone or in combination, in the presence or absence of serum or albumin was studied. There was strong synergy between the cytokines in inducing human umbilical vein endothelial cell apoptosis, but only in the absence of serum. Synergy was destroyed by boiling cytokines and was not affected by polymyxin B. Dose response experiments revealed greater activity of TGF-beta(1) than TGF-beta(2). The synergy was protein synthesis dependent and apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and FACS analysis. Data suggests a role for synergistic activation of endothelial cell apoptosis by TNF-alpha and TGF-beta(1) but perhaps only in poorly perfused vessels deprived of serum factors.