Blood Fibrocytes Research Articles

Circulating fibrocyte levels correlate with left ventricular mass in middle-aged healthy adults without hypertension

BackgroundFibrocytes, circulating bone-marrow derived cells that differentiate into fibroblasts and myofibroblasts, are a major source of hypertensive arterial fibrosis and correlate with left ventricular (LV) mass in subjects with hypertension. We tested whether circulating fibrocytes levels correlate with LV mass in middle-aged adults without hypertension. MethodsWe measured peripheral blood fibrocyte levels and their activated phenotypes in 13 middle-aged, non-hypertensive adults and performed cardiac magnetic resonance imaging to assess LV mass. ResultsThere was a strong correlation between total fibrocyte levels (CD45 + Col1+) and LV mass index (r = 0.71, p = 0.006), as well as fibrocyte subsets expressing the chemokine markers CCR2 (r = 0.60, p = 0.032), CCR5 (r = 0.62, p = 0.029), CCR7 (r = 0.60, p = 0.034), co-expression of CXCR4 and CCR2 (r = 0.62, p = 0.029), α-SMA+ (r = 0.57, p = 0.044), CD133 (r = 0.59, p = 0.036), and pSTAT6 (r = 0.64, p = 0.032). ConclusionsCirculating fibrocytes are associated with LV mass index in middle-aged, non-hypertensive adults and may be a harbinger for the development of hypertension.

Characterization of Circulating Fibrocytes in People Living with HIV on Stable Antiretroviral Therapy.

Highly effective combination antiretroviral therapy has reduced HIV infection to a manageable chronic disease, shifting the clinical landscape toward management of noninfectious comorbidities in people living with HIV (PLWH). These comorbidities are diverse, generally associated with accelerated aging, and present within multiple organ systems. Mechanistically, immune dysregulation and chronic inflammation, both of which persist in PLWH with well-controlled virally suppressive HIV infection, are suggested to create and exacerbate noninfectious comorbidity development. Persistent inflammation often leads to fibrosis, which is the common end point pathologic feature associated with most comorbidities. Fibrocytes are bone marrow-derived fibroblast-like cells, which emerged as key effector cells in tissue repair and pathologic fibrotic diseases. Despite their relevance to fibrosis, the circulating fibrocyte concentration in PLWH remains poorly characterized, and an understanding of their functional role in chronic HIV is limited. In this study, utilizing PBMCs from a cross-sectional adult HIV cohort study with matched uninfected controls (HIV-), we aimed to identify and compare circulating fibrocytes in blood. Both the percentage and number of fibrocytes and α-smooth muscle actin+ fibrocytes in circulation did not differ between the HIV+ and HIV- groups. However, circulating fibrocyte levels were significantly associated with increasing age in both the HIV+ and HIV- groups (the percentage and number; r = 0.575, p ≤ 0.0001 and r = 0.558, p ≤ 0.0001, respectively). Our study demonstrates that circulating fibrocyte levels and their fibroblast-like phenotype defined as collagen I and α-smooth muscle actin+ expression are comparable between, and strongly associated with, age irrespective of HIV status.

Circulating and renal fibrocytes are associated with interstitial fibrosis in lupus nephritis.

Fibrocytes, the extracellular matrix-producing cells derived from bone marrow progenitors, contribute to organ fibrosis. We investigated the presence and characteristics of fibrocytes in the peripheral blood and kidney of patients with lupus nephritis (LN), and the association of the abundance of fibrocytes with renal tubular epithelial cells (RTECs) in LN fibrogenesis. Fibrocytes were identified with type I collagen (colI), α-smooth muscle actin (α-SMA), CD34 and CD45 using flow cytometry and confocal imaging. The associations between the levels of fibrocytes and pathological features of patients with LN were analysed. The contribution of RTECs to fibrocyte generation was determined using LN sera-treated HK-2 cells. Spindle-shaped fibrocytes (colI+α-SMA+CD34+CD45+ cells) were present in the peripheral blood and their abundance was especially high in LN patients with interstitial fibrosis compared with healthy control. Renal fibrocytes (colI+α-SMA+CD45+ cells) were found in the tubulointerstitium in patients with LN, and their numbers were significantly associated with the degrees of chronicity indices including interstitial fibrosis and renal dysfunction. Stimulation of peripheral blood mononuclear cells with supernatants from LN serum-treated HK-2 cells led to a significant generation of fibrocytes, which was abrogated by the addition of IL-6 neutralizing antibody. Fibrocytes were significantly increased in the blood and kidney tissue of patients with LN, especially those with interstitial fibrosis. Fibrocytes could be differentiated from blood cells, with an active contribution from RTECs. Our results show a possible link between fibrocytes and tubulointerstitial fibrosis, which may serve as a novel therapeutic target for LN fibrogenesis.

Oxygen Desaturation Is Associated With Fibrocyte Activation via Epidermal Growth Factor Receptor/Hypoxia-Inducible Factor-1α Axis in Chronic Obstructive Pulmonary Disease.

Fibrocytes are bloodborne mesenchymal progenitors which accumulate and differentiate at the disease site. We investigated whether hypoxemia activates fibrocytes, accelerating airflow limitation and exercise intolerance in chronic obstructive pulmonary disease (COPD) patients. Flow cytometry was used to determine collagen I+/CD45+ fibrocytes and α-smooth muscle actin+ differentiating fibrocytes within peripheral blood and cultured cells, as well as the expression of CXC chemokine receptor 4 (CXCR4), epidermal growth factor receptor (EGFR), connective tissue growth factor (CTGF) and hypoxia-inducible factor (HIF)-1α. Fibrocytes in lung specimens were identified by confocal microscopy. Compared to non-desaturators, COPD desaturators (peripheral blood oxygen saturation ≤88% during exercise) had greater number of fibrocytes in peripheral blood and lung specimens, paralleled with faster yearly lung function decline and a 6-minute walk distance. Fibrocytes from desaturators expressed more EGFR, CXCR4, CTGF, and HIF-1α, with a higher capacity of proliferation and myofibroblastic differentiation. Hypoxia (5% oxygen) increased the expression of EGFR, CXCR4, CTGF, and HIF-1α, the number and differentiation in fibrocytes. These effects were attenuated by EGFR inhibitor gefitinib, HIF-1α gene silencing, and anti-CTGF antibody. These data elucidate that hypoxemia triggers fibrocyte activation through the EGFR/HIF-1α axis, aggravating airflow obstruction in COPD.

Comparison of circulating fibrocytes from non-asthmatic patients with seasonal allergic rhinitis between in and out of pollen season samples

BackgroundAllergic rhinitis is a risk factor for asthma development. In asthma, fibroblast progenitors, fibrocytes, are increased in the blood and bronchial mucosa following allergen exposure. These cells may play a role in lower airways remodeling as observed in non-asthmatic subjects with allergic rhinitis.ObjectiveTo determine the influence of seasonal allergen exposure on blood circulating fibrocytes in allergic rhinitic subjects without asthma.MethodsNon-asthmatic subjects with seasonal allergic rhinitis had blood sampling at baseline and at the peak of rhinitis symptoms. Cells were stained for fibrocyte markers (CD34, CD45, CXCR4, collagen I) and analyzed by flow cytometry.ResultsData from 26 subjects (11M:15F) aged 29 ± 8 years were analysed. Compared to baseline, there was a significant decrease in blood fibrocytes during the pollen season in subjects sensitized to trees [median (25–75 percentile), 9.3 (6.4–20.7)% vs 7.0 (4.2–10.1)%, P = 0.007] and a significant increase in subjects sensitized to grass [12.7 (9.9–23.1)% vs 64.0 (57.6–73.6)%, P < 0.001] and ragweed [8.0 (7.4–10.8)% vs 48.2 (43.5–52.6)%, P < 0.001]. A significant decrease in CXCR4 mean fluorescence was also observed between the two visits [1814 (1261–2235) vs 1352 (814–1796) (arbitrary units), P = 0.02].Conclusions and clinical relevanceThese results contribute to document dynamic variations in blood fibrocytes’ activation and migration into the airways following natural exposure to allergens. These findings may help identify one of the potential factors involved in the development of asthma in allergic rhinitic subjects.

Role of Circulating Hematopoietic Fibrocytes in Chronic Hepatitis C Patients Induced Liver Fibrosis

Background: Bone marrow derived fibrocytes may play an important role in pathogenesis and resolution of liver fibrosis. These cells may offer new approaches for better understanding the pathogenesis of liver fibrosis.&#x0D; Aim of the work: To define the proportion of circulating fibrocytes with hematopoietic progenitor origin as defined by CD45 and CD34 positivity and to assess whether they are increased in patients with chronic C hepatitis in correlation to the degree of liver fibrosis.&#x0D; Subjects and Methods: Sixty HCV patients were classified according to METAVIR score into 4 stages of liver fibrosis, 15 age and sex-matched controls were included. Flowcytometric analysis for circulating levels of fibrocytes CD34+ve cells, CD45+ve cells, collagen type I+ve cells and CXCR4+ve cells was carried out using monoclonal antibodies (anti-CD34, CD45, collagen type I and CD184). GM-CSF, TGF-β and α-SMA were assessed using ELISA.&#x0D; Results and Conclusions: A significant increase in the circulating levels of GM-CSF, TGF- β and α-SMA, with a significant increase in the percentage of cells express CXCR4and in the co expression of CD34, CD45 and collagen type I positive cells in different groups of patients compared to control group, denoting the presence of an increased proportion of circulating fibrocytes in peripheral blood of these patients. The percentage of fibrocytes that positively expression CD34, CD45, collagen type I and CXCR4, were increased in step wise fashion in conjunction with worsening severity of liver disease.&#x0D; Liver fibrosis is associated with increased levels of circulating TGF-β1 and lipopolysaccharide, activation of myofibroblasts, and extensive deposition of extracellular matrix, mostly collagen Type I. TGF-β and LPS play a critical role in fibrogenesis and trigger fibrocyte recruitment to the injured liver promoting their differentiation into collagen type I producing myofibroblast, supporting that fibrocytes may become a novel target for anti fibrotic therapy.

Circulating fibrocyte levels correlate with infarct size in patients with ST elevation myocardial infarction treated with primary percutaneous coronary intervention

Study objectiveInfarct size is a strong predictor of outcomes after ST elevation myocardial infarction (STEMI). Circulating fibrocytes are bone marrow-derived progenitor cells associated with fibrotic processes. We tested whether fibrocytes correlate with infarct size in STEMI patients treated with primary percutaneous coronary intervention (PCI). DesignProspective observational study. SettingAcademic medical center. ParticipantsSubjects with STEMI treated with primary PCI. InterventionsPeripheral blood draw and cardiac magnetic resonance imaging (CMR). Main outcome measureCorrelation of fibrocyte levels with infarct size. MethodsPeripheral blood fibrocytes were quantified at discharge from STEMI hospitalization and at 6 months follow-up using flow cytometry. Infarct size was determined within 2 weeks of discharge and at 6 months follow-up using late gadolinium enhancement on CMR. ResultsAmong 14 patients (median age 54 years, 79% men) with STEMI, there was a statistically significant positive correlation between fibrocyte levels at 6 months and 6-month infarct size on CMR (r = 0.58, p = 0.031). In addition, there was positive correlation between peak troponin I level (r = 0.85, p < 0.001), and white blood cell count (r = 0.55, p = 0.042) during the hospital stay and 6-month infarct size on CMR. ConclusionsCirculating fibrocytes measured 6 months after STEMI positively correlate with 6-month infarct size assessed by CMR.

Peripheral blood mononuclear cells - Can they provide a clue to the pathogenesis of Graves' Orbitopathy?

Graves' disease (GD) is an autoimmune disorder affecting primarily the thyroid gland. The most common extrathyroidal manifestation of GD is known as Graves' orbitopathy (GO). Bone marrow-derived fibrocytes represent a subset of monocytes in peripheral blood mononuclear cells (PBMCs), infiltrate the orbital tissues, and contribute to the pathogenesis of GO. Hence objectives of the study included whether the concentration of fibrocytes in peripheral blood was higher in GO, whether TSHR m RNA expression and TSHR surface expression in peripheral blood were higher in GO in comparison to Graves' Disease (GD) and Control subjects. The percentage of circulating fibrocytes (FC) along with TSHR on its cell surface (CD 34+, CD 45+, CXCR4+, Collagen 1+, TSHR+) were assessed by flow cytometry of 50 patients with GD and GO and 15 healthy donors (Control). TSHR mRNA expression was measured by q RT PCR. The concentration of circulating fibrocytes was significantly higher in GO compared to GD and control [GO 17% vs GD 3% vs control 0.7% (p < 0.05)]. Moreover, these fibrocytes express a significantly higher level of TSHR in GO. This was corroborated by the measure of TSH mRNA; in GD it was 2.3-fold higher and in GO it was 3.9 fold higher than in control, in GO this transcript level was 1.7fold higher than GD (p < 0.05). TSHR+ fibrocytes were significantly positively correlated with CAS (p = 0.004) and negatively correlated with age (p = 0.01) and duration of disease (p = 0.01) in GO. This study sheds further light on the pathogenesis of GO.

Cofilin-1 promotes fibrocyte differentiation and contributes to pulmonary fibrosis

Fibrocytes originate from the bone marrow monocyte lineage and participate in the pathogenesis of pulmonary fibrosis. Research providing a comprehensive picture of fibrocytes is still limited. Cofilin-1 (CFL-1) is an important protein that regulates cell proliferation, migration and differentiation. Whether CFL-1 can induce monocyte differentiation into fibrocytes and promote the process of pulmonary fibrosis is unknown. Compared with that of healthy controls, the expression of CFL-1 was significantly increased in the plasma and peripheral blood mononuclear cells (PBMCs) from idiopathic pulmonary fibrosis (IPF) and connective tissue disease-associated interstitial lung disease (CTD-ILD) patients (P < 0.05). The percentages of peripheral blood fibrocytes in the IPF group (4.2550 ± 0.3483%) and CTD-ILD group (4.7100 ± 0.4811%) were higher than that in the control group (1.6340 ± 0.2549%) (both P < 0.05). In vitro, PBMCs transfected with siRNA-CFL-1 showed lower expression of CFL-1, and the percentage of fibrocytes was lower than that of the control (P < 0.05). PBMCs transfected with Lv-CFL-1 to increase the expression of CFL-1 showed a higher percentage of fibrocytes than the control (P < 0.05). In mice with bleomycin-induced pulmonary fibrosis, the relative expression of CFL-1 was increased, and the percentage of fibrocytes was higher than that in the saline group (P < 0.05). In bleomycin-induced mice, interference with Lv-CFL-1 decreased the expression of CFL-1, the percentage of fibrocytes was lower, and the lung tissue showed less fibrosis (P < 0.05). The overexpression of CFL-1 is associated with pulmonary fibrogenesis. CFL-1 could promote the differentiation of fibrocytes from monocyte peripheral blood mononuclear cells and promote pulmonary fibrosis.

Fibrocytes in early and long-standing rheumatoid arthritis: a 6-month trial with repeated synovial biopsy, imaging and lung function test

ObjectivesTo correlate the level of fibrocytes in peripheral blood, synovial tissue and in vitro culture in rheumatoid arthritis (RA) with changes in disease activity, imaging and pulmonary function.MethodsTwenty patients with...

Oxygen Desaturation Is Associated With Fibrocyte Activation via Epidermal Growth Factor Receptor/ Hypoxia-Inducible Factor-1α Axis in Chronic Obstructive Pulmonary Disease

Background: Fibrocytes are bloodborne mesenchymal progenitors which accumulate and differentiate at disease site. We investigated whether hypoxemia activates fibrocytes, accelerating airflow limitation and exercise intolerance in chronic obstructive pulmonary disease (COPD) patients. Methods: Flow cytometry was used to determine collagen I+/CD45+ fibrocytes and α-smooth muscle actin+ differentiating fibrocytes within peripheral blood and cultured cells, as well as the expression of CXC chemokine receptor 4 (CXCR4), epidermal growth factor receptor (EGFR), connective tissue growth factor (CTGF) and hypoxia-inducible factor (HIF)-1α. Fibrocytes in lung specimens were identified by confocal microscopy. Findings: Compared to non-desaturators, COPD desaturators (peripheral blood oxygen saturation ≤88% during exercise) had greater number of fibrocytes in peripheral blood and lung specimens, paralleled with faster yearly lung function decline and 6-minute walk distance. Fibrocytes from desaturators expressed more EGFR, CXCR4, CTGF and HIF-1α, with a higher capacity to proliferation and myofibroblastic differentiation. Hypoxia (5% oxygen) increased the expression of EGFR, CXCR4, CTGF and HIF-1α, the number and differention in fibrocytes. These effects were attenuated by EGFR inhibitor gefitinib, HIF-1α gene silencing, and anti-CTGF antibody. Interpretation: These data elucidate that hypoxaemia triggers fibrocyte activation through the EGFR/HIF-1α axis, aggravating airflow obstruction in COPD. Funding Information: This work was supported by the National Medical Research Program (NMRP) grant 99-2314-B-182-043-MY3, 108-2314-B-182A-128-MY2 and the Chang Gung Medical Research Project (CMRP) grant CMRPG390041-2. Declaration of Interests: The authors have declared that no competing interest exists. Ethics Approval Statement: The study was approved by the Ethics Committee of Chang Gung Memorial Hospital (IRB: 98-3950B, 201801979A3). Written informed consents were obtained from all participants.

Neutrophil Extracellular Traps Induce MCP-1 at the Culprit Site in ST-Segment Elevation Myocardial Infarction.

BackgroundLeukocyte-mediated inflammation is crucial in ST-segment elevation myocardial infarction (STEMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in STEMI.MethodsCulprit site and peripheral blood samples of STEMI patients were drawn during primary percutaneous coronary intervention. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA while double-stranded DNA was stained with a fluorescent dye. The influence of MCP-1 on NET formation in vitro was assessed using isolated healthy donor neutrophils. Human coronary artery endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 gene expression was measured by ELISA and qPCR. CCR2 expression of culprit site and peripheral blood fibrocytes was characterized by flow cytometry. Healthy donor fibrocyte receptor expression and chemotaxis were investigated in response to stimulation with MCP-1 and NETs in vitro.ResultsNETs and concentrations of MCP-1 were increased at the culprit site of 50 consecutive STEMI patients. NET stimulation of hCAECs induced transcription of ICAM-1, IL-6, and MCP-1, and secretion of MCP-1. MCP-1 promoted NET formation of healthy donor neutrophils in vitro. An increasing MCP-1 gradient correlated with fibrocyte accumulation at the culprit site. Locally increased MCP-1 levels were negatively correlated with CCR2 expression on fibrocytes. MCP-1 and NETs induced CCR2 downregulation on fibrocytes in vitro. NETs did not function as a chemotactic stimulus for fibrocytes or monocytes and could block migration in response to MCP-1 for both cell populations.ConclusionNETs function as signaling scaffolds at the culprit site of STEMI. NETs assist MCP-1 and ICAM-1 release from culprit site coronary artery endothelial cells. MCP-1 facilitates further NETosis. Monocytes enter the culprit site along an MCP-1 gradient, to transdifferentiate into fibrocytes in the presence of NETs.

Fibrotic remodeling of left ventricle in the murine remnant kidney model of chronic kidney disease is independent of arterial hypertension

Abstract Background Cardiac fibrosis and arterial hypertension are common in patients with chronic kidney disease (CKD). We studied the mechanisms of cardiac fibrogenesis and the role of blood pressure in mice with CKD. Methods and results 10-week-old male C57/BL6N (BL6) and SV129 wildtype (WT) mice were underwent 5/6 nephrectomy (remnant kidney model, RKM) or sham operation for 10 weeks. RKM significantly elevated plasma creatinine and urea. RKM elicited both interstitial and replacement renal and left ventricular (LV) (BL6: SHAM 5.6±0.4%, RKM 7.3±0.7%, p=0.04; SV129: SHAM 6.0±0.5%, RKM 14±2%, p=0.001) fibrosis as assessed by picrosirius red staining. In parallel, the number of cardiac fibroblasts per mm2 (BL6: SHAM 36±4, RKM 85±13, p=0.001; SV129: SHAM 82±11, RKM 200±34, p=0.006) was increased in RKM mice. With regard to possible mechanisms, cardiac oxidative stress as shown by co-immunostaining for intracellular fibronectin and 8-hydroxyguanosine (BL6: SHAM 44±14%, RKM 60±24%, p=0.03; SV129: SHAM 51±6%, RKM 70±7%, p=0.04) and the percentage of CXCR4+ fibroblasts in the myocardium (BL6: SHAM 47±5%, RKM 62±4%, p=0.04; SV129: SHAM 63±5%, RKM 81±3%, p=0.005) were increased. Furthermore, the number of circulating CD45+ / collagen I+ fibrocytes (FACS) in the peripheral blood was increased by RKM in BL6 (SHAM 100±23%, RKM 443±252%, p=0.04) and diminished in SV129 (SHAM 100±19%, RKM 43±11%, p=0.01), while an opposite regulation was seen in the bone marrow. To further confirm the role of bone-marrow derived fibroblasts in renal and cardiac remodeling 10-week-old WT BL6 mice were subjected to transplantation of bone marrow from 10-week-old WT BL6 mice expressing green fluorescent protein (GFP)+ ubiquitously. 28 days later, RKM or SHAM-operation was performed. RKM significantly increased the number of GFP+ fibroblasts in kidney and LV-myocardium. CKD significantly decreased myocardial capillarization assessed by immunostaining for podocalyxin in both mouse lines. In parallel, myocardial protein expression of fibrosis regulators fibronectin, collagen I, CTGF and Hif1a were up-regulated and expression of the active form of eNOS (phospho-S1177) was reduced. As a possible confounder, tail-cuff blood pressure was moderately enhanced (Ø 30mmHg) 9 weeks after nephrectomy. In a control experiment using the vasodilator hydralazine (250 mg/L/day), peripheral blood pressure was equalized in all 4 experimental groups, but the extent of LV fibrosis and expression of the above-mentioned fibrosis markers remained unchanged. Conclusions Chronic kidney disease in the RKM model elicits left ventricle fibrosis by increasing myocardial protein expression of fibrosis regulators, reduction of myocardial capillarization and mobilization / recruitment of circulating fibroblasts, independently of blood pressure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft

Circulating fibrocytes as prognostic biomarkers of autoimmune interstitial lung disease.

BackgroundAutoimmunity is a common cause of pulmonary fibrosis and can present either as a manifestation of an established connective tissue disease or as the recently described entity of interstitial pneumonia with autoimmune features. The rate of progression and responsiveness to immunosuppression in these illnesses are difficult to predict. Circulating fibrocytes are bone marrow-derived progenitor cells that home to injured tissues and contribute to lung fibrogenesis. We sought to test the hypothesis that the blood fibrocyte concentration predicts outcome and treatment responsiveness in autoimmune interstitial lung diseases.MethodsWe compared the concentration of circulating fibrocytes in 50 subjects with autoimmune interstitial lung disease and 26 matched healthy controls and assessed the relationship between serial peripheral blood fibrocyte concentrations and clinical outcomes over a median of 6.25 years.ResultsAs compared to controls, subjects with autoimmune interstitial lung disease had higher circulating concentrations of total fibrocytes, the subset of activated fibrocytes, and fibrocytes with activation of PI3K/AKT/mTOR, transforming growth factor-β (TGF-β) receptor and interleukin (IL)-4/IL-13 receptor signalling pathways. Over the follow-up period, there were episodes of marked elevation in the concentration of circulating fibrocytes in subjects with autoimmune interstitial lung disease but not controls. Initiation of immunosuppressive therapy was associated with a decline in the concentration of circulating fibrocytes. For each 100 000 cells·mL−1 increase in peak concentration of circulating fibrocytes, we found a 5% increase in odds of death or lung function decline.ConclusionIn patients with autoimmune interstitial lung disease, circulating fibrocytes may represent a biomarker of outcome and treatment response.

Circulating fibrocytes traffic to the lung in murine acute lung injury and predict outcomes in human acute respiratory distress syndrome: a pilot study

BackgroundFibrosis is an integral component of the pathogenesis of acute lung injury and is associated with poor outcomes in patients with acute respiratory distress syndrome (ARDS). Fibrocytes are bone marrow-derived cells that traffic to injured tissues and contribute to fibrosis; hence their concentration in the peripheral blood has the potential to serve as a biomarker of lung fibrogenesis. We therefore sought to test the hypothesis that the concentration and phenotype of circulating fibrocytes in patients with ARDS predicts clinical outcomes.MethodsFor the animal studies, C57Bl/6 mice were infected with experimental Klebsiella pneumoniae in a model of acute lung injury; one-way ANOVA was used to compare multiple groups and two-way ANOVA was used to compare two groups over time. For the human study, 42 subjects with ARDS and 12 subjects with pneumonia (without ARDS) were compared to healthy controls. Chi-squared or Fisher’s exact test were used to compare binary outcomes. Survival data was expressed using a Kaplan-Meier curve and compared by log-rank test. Univariable and multivariable logistic regression were used to predict death.ResultsIn mice with acute lung injury caused by Klebsiella pneumonia, there was a time-dependent increase in lung soluble collagen that correlated with sequential expansion of fibrocytes in the bone marrow, blood, and then lung compartments. Correspondingly, when compared via cross-sectional analysis, the initial concentration of blood fibrocytes was elevated in human subjects with ARDS or pneumonia as compared to healthy controls. In addition, fibrocytes from subjects with ARDS displayed an activated phenotype and on serial measurements, exhibited intermittent episodes of markedly elevated concentration over a median of 1 week. A peak concentration of circulating fibrocytes above a threshold of > 4.8 × 106 cells/mL cells correlated with mortality that was independent of age, ratio of arterial oxygen concentration to the fraction of inspired oxygen, and vasopressor requirement.ConclusionsCirculating fibrocytes increase in a murine model of acute lung injury and elevation in the number of these cells above a certain threshold is correlated with mortality in human ARDS. Therefore, these cells may provide a useful and easily measured biomarker to predict outcomes in these patients.

Histological Examination of the Osteogenic Properties of White Acropora Coral Grainules Sized 50–150 μm Under Subcutaneous Administration in the Experiment

The aim of research was to study morphological characteristics of the osteogenic properties of the natural white Acropora coral skeleton granules when implanted under the skin.Material and methods. The study included 60 sexually mature male Wistar rats. The object of the morphological study was subcutaneous implants isolated for different periods of the experiment. The experimental animals were divided into three groups, 20 animals each. In the 1st and 2nd groups the granules were placed in a gelatin capsule, in the 3rd group – the granules were implanted directly under the skin of the animals. When preparing implants for the 1st group, the granules were treated with the blood of an animal taken from the tail vein during surgery. Implants for the 2nd group of animals were not treated with blood. Histological research methods (hematoxylin and eosin staining according to Mallory, Weigert–Van Gieson, and Masson–Goldner; staining to determine the age of fibrin (BioVitrum)) were used in the study; the results were statistically processed.Results. The results obtained demonstrated that heterotopic bone formation based on granules from the skeleton of natural coral was only possible when they were in wide contact with blood on the first day of the experiment. The results of the study also evidenced that blood clot elements, fibrocytes, and osteoblasts were the source for the development of newly formed bone tissues. The gelatin capsule allowed temporarily restricting coral granules from the surrounding tissues with peripheral venous blood cells inside; the fact contributing to the formation and accumulation of cellular elements and subsequently leading to the formation of young bone tissues.Conclusions. As demonstrated, subcutaneous implantation of coral granules in a gelatin capsule without blood did not result in osteogenesis. Due to the apparent osteoinductive potential, natural coral skeleton granules applied according to the proposed method are considered to be an alternative to other osteoplastic materials for the stimulation of de novo bone formation.

Blood Fibrocytes During an Exacerbation and Lung Function Decline in Patients With COPD in Primary Care.

Blood Fibrocytes During an Exacerbation and Lung Function Decline in Patients With COPD in Primary Care.

Fibrocytes are Increased in the Medial Layer of the Aorta from Spontaneously Hypertensive Rats

BackgroundChronic hypertension causes a state of inflammation and leads to vascular remodeling. Vascular remodeling involves alterations in vascular smooth muscle cells, endothelium, and extracellular matrix. Collagen deposition is increased in conductance and resistance vessels of hypertensive animals.Fibrocytes are bone marrow‐derived cells found in the circulation that migrate toward injured tissue. At the injured site, fibrocytes contribute to healing by serving as antigen presenting cells and secrete cytokines such as IL‐6 and IL‐8 to recruit T cells. Fibrocytes also lay down extracellular matrix such as collagen, and its activation is implicated in many fibrotic diseases including cardiac fibrosis. Studies have shown that patients with hypertensive heart disease have increased number of circulating fibrocytes and that hearts of hypertensive patients have increased collagen levels. The role of fibrocytes in vascular remodeling in hypertension have not been investigated.HypothesisThis study tests the hypothesis that fibrocytes are increased in hypertension and play a role in vascular remodeling.MethodsMale spontaneously hypertensive rats (SHR) and Wistar rats 13–16 weeks old were used in this study. Flow cytometry was used to determine the percentage of fibrocytes (CD34+/CD45+/CollagenI+) in the peripheral blood, aorta, mesenteric resistance arteries (MRA), and pudendal arteries. Wire myograph was used to evaluate the differences in vascular function in response to phenylephrine (PE), norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP) between SHR and Wistar. Immunohistochemistry was used to detect fibrocytes (CD34+/CD45+/Collagen I+) within the vascular walls of aorta, mesenteric, and pudendal arteries. Statistical analysis performed using nonlinear regression, two‐tailed unpaired t‐test, and two‐way ANOVA.ResultsFlow cytometry did not show a difference between the percentage of fibrocytes in SHR compared to Wistar. Wire myography demonstrated differences between the two groups in aortic contraction to PE (p&lt;0.0001) and NE (p&lt;0.0001), aortic relaxation to ACh (p&lt;0.0001) and SNP (p&lt;0.0001), MRA relaxation to ACh (p&lt;0.0001), and pudendal artery relaxation to SNP (p&lt;0.0001). Immunohistochemistry showed increased number of fibrocytes in the media layer of the SHR aorta (Figure 1) compared to Wistar (Figure 2).ConclusionsThe number of fibrocytes are increased in the media layer in the aorta in SHR compared to Wistar. The increased number of fibrocytes may contribute to increased collagen deposition leading to decreased vascular compliance in hypertensive animals.Support or Funding InformationNIH P01 HL‐134604This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Raf kinase inhibitor protein mediates myocardial fibrosis under conditions of enhanced myocardial oxidative stress

Fibrosis is a hallmark of maladaptive cardiac remodelling. Here we report that genome-wide quantitative trait locus (QTL) analyses in recombinant inbred mouse lines of C57BL/6 J and DBA2/J strains identified Raf Kinase Inhibitor Protein (RKIP) as genetic marker of fibrosis progression. C57BL/6 N-RKIP−/− mice demonstrated diminished fibrosis induced by transverse aortic constriction (TAC) or CCl4 (carbon tetrachloride) treatment compared with wild-type controls. TAC-induced expression of collagen Iα2 mRNA, Ki67+ fibroblasts and marker of oxidative stress 8-hydroxyguanosine (8-dOHG)+ fibroblasts as well as the number of fibrocytes in the peripheral blood and bone marrow were markedly reduced in C57BL/6 N-RKIP−/− mice. RKIP-deficient cardiac fibroblasts demonstrated decreased migration and fibronectin production. This was accompanied by a two-fold increase of the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), the main transcriptional activator of antioxidative proteins, and reduced expression of its inactivators. To test the importance of oxidative stress for this signaling, C57BL/6 J mice were studied. C57BL/6 J, but not the C57BL/6 N-strain, is protected from TAC-induced oxidative stress due to mutation of the nicotinamide nucleotide transhydrogenase gene (Nnt). After TAC surgery, the hearts of Nnt-deficient C57BL/6 J-RKIP−/− mice revealed diminished oxidative stress, increased left ventricular (LV) fibrosis and collagen Iα2 as well as enhanced basal nuclear expression of Nrf2. In human LV myocardium from both non-failing and failing hearts, RKIP-protein correlated negatively with the nuclear accumulation of Nrf2. In summary, under conditions of Nnt-dependent enhanced myocardial oxidative stress induced by TAC, RKIP plays a maladaptive role for fibrotic myocardial remodeling by suppressing the Nrf2-related beneficial effects.

Crystalline Silica Promotes Rat Fibrocyte Differentiation in Vitro, and Fibrocytes Participate in Silicosis in Vivo

ObjectiveThe aim of this study was to investigate the effects of SiO2 on fibrocytes and whether fibrocytes participate in silicosis in vivo. MethodsA macrophagocyte (AM)/fibrocyte coculture system was established, and AMs were treated with 100 µg/mL SiO2. Flow cytometry was used to detect the number of fibrocytes. Real-time PCR was performed to measure the expression of collagen I, collagen III, and α-SMA mRNA. The levels of collagen I, collagen III, and TGF-β1 protein were determined by ELISA. Immunohistochemical staining was performed to measure α-SMA protein expression. A rat silicosis model was induced by intratracheal instillation of SiO2. Lung histopathological evaluation was conducted using HE and Masson's trichrome staining after 1 and 9 weeks. The number of fibrocytes in peripheral blood or lung tissue of rat was detected by flow cytometry. Double-color immunofluorescence was applied to identify fibrocytes in the lung tissue. ResultsPeripheral blood monocytes were found to differentiate into fibrocytes in vitro in a time-dependent manner, and exposure to crystalline silica might potentiate fibrocyte differentiation. In addition, fibrocytes were able to migrate from peripheral blood to the lung tissue, and the number of fibrocytes was increased after SiO2 exposure. ConclusionSilica exposure potentiates fibrocyte differentiation, and fibrocytes may participate in silicosis in vivo.