Number Of Fibrocytes Research Articles

Fibrocytes and Fibrovascular Membrane Formation in Proliferative Diabetic Retinopathy.

The purpose of this study was to investigate whether fibrocytes participate in formation of the fibrovascular membrane (FVM) in patients with proliferative diabetic retinopathy (PDR). Vitreous fluid and FVM samples were obtained during vitrectomy in patients with PDR. Samples from patients with macular hole or epiretinal membrane were used as controls. Vitreous fluid and FVM samples were subjected to immunohistochemical analysis. In addition, cells isolated from the vitreous fluid of PDR and control patients were cultured in serum-free medium. Fibrocytes were identified among these cells by morphological and immunohistochemical analyses. We examined the number of fibrocytes in PDR patients and control patients. Also, the concentrations of monocyte chemoattractant protein-1 (MCP-1), pentraxin3, and serum amyloid P (SAP) in vitreous fluid samples from PDR patients and control patients were determined by enzyme-linked immunosorbent assay. Fibrocytes were observed in the vitreous and FVM samples from PDR patients. Cells cultured from the vitreous samples of PDR patients were spindle shaped and expressed fibrocyte markers. TGF-β1 induced differentiation of these cells into myofibroblasts. The number of fibrocytes was higher in samples from PDR patients than in samples from control patient. The vitreous fluid concentration of MCP-1 was significantly higher in PDR patients than in controls and showed a significant positive correlation with the number of fibrocytes from the vitreous fluid. Vitreous fluid concentrations of pentraxin3 and SAP were also higher in PDR patients than in control patients. These findings indicate that fibrocytes may be involved in development of the FVM in PDR.

Circulating Fibrocytes Are Increased in Neonates with Bronchopulmonary Dysplasia.

BackgroundBronchopulmonary dysplasia (BPD) is characterized by the aberrant remodeling of the lung parenchyma, resulting from accumulation of fibroblasts or myofibroblasts. Circulating fibrocytes are implied in pulmonary fibrosis, but whether these cells are associated with the development of BPD or the progressive fibrosis is unknown. The aim of the present study was to investigate the occurrence of fibrocytes in peripheral venous blood and explore whether these cells might be associated with severity of BPD.MethodsWe investigated circulating fibrocytes in 66 patients with BPD, 23 patients with acute respiratory distress syndrome(ARDS) and 11 normal subjects. Circulating fibrocytes were defined and quantified as cells positive for CD45 andcollagen-1 by flow cytometry. Furthermore, serum SDF-1/CXCL12 and TGF-β1 were evaluated using ELISA methods. We also investigated the clinical value of fibrocyte counts by comparison with standard clinical parameters.ResultsThe patients with BPD had significantly increased numbers of fibrocytes compared to the controls (p < 0.01). Patients with ARDS were not different from healthy control subjects. There was a correlation between the number of fibrocytes and pulmonary hypertension or oxygen saturation (p < 0.05). Fibrocyte numbers were not correlated with other clinical or functional variables or radiologic severity scores. The fibrocyte attractant chemokine CXCL12 increased in plasma (p < 0.05) and was detectable in the bronchoalveolar lavage fluid of 40% of the patients but not in controls.ConclusionThese findings indicate that circulating fibrocytes are increased in patients with BPD and may contribute to pulmonary fibrosis in BPD. Circulating fibrocytes, likely recruited through the CXCR4/CXCL12 axis, might contribute to the production of TGF-β1 for the expansion of fibroblast/myofibroblast population in BPD.

Imatinib ameliorates bronchiolitis obliterans via inhibition of fibrocyte migration and differentiation

Imatinib, a tyrosine kinase inhibitor, has been proposed as a potential anti-fibrotic agent for fibroproliferative diseases, including bronchiolitis obliterans (BO). However, the underlying anti-fibrotic mechanisms of the agent remain unclear. We evaluated whether bone (BM)-derived progenitor cells, fibrocytes, might be a target of imatinib in the attenuation of BO. We used a murine BO model induced by heterotopic tracheal transplantation and assessed the origin of fibroblasts by using green fluorescent protein-BM chimeric mice. We also evaluated the effects of imatinib on luminal obstruction and fibrocyte accumulation. The effects of imatinib on fibrocyte migration and differentiation were assessed by culturing fibrocytes in vitro. In the murine BO model, tracheal allografts showed epithelial injury and developed complete luminal occlusion 28 days after transplantation. Most of the mesenchymal cells that had accumulated in the tracheal allograft were derived from BM cells. Imatinib treatment ameliorated the airway luminal occlusion and significantly reduced the number of fibrocytes in the allografts. In vitro studies showed that imatinib inhibited migration of cultured blood fibrocytes via the platelet-derived growth factor/platelet-derived growth factor receptor axis. Imatinib also inhibited differentiation of fibrocytes via suppression of c-Abl activity that was essential for the differentiation of monocytes to fibrocytes. Imatinib prevents airway luminal obstruction by inhibiting the migration and differentiation of fibrocytes. Fibrocytes may be a novel target in the prevention and treatment of BO.

Mechanism of Atrial Fibrillation is Related to Changes of Type IV Collagen in Rheumatic Heart Valvular Disease

PurposeAtrial fibrillation (AF) is a common complication in heart valvular disease and is associated with morphology and cell ultrastructure abnormality. We aimed to study the distribution of collagen types and to investigate the relationship between type IV collagen changes in the tissue of the right atrium and the development of permeant AF in patients with mitral valvular disease.MethodsThe atrial appendage obtained from patients with mitral valvular disease associated with either sinus rhythm (SR, n=26, right atrial tissue) or permanent AF (AF ≥6 months, n=36, right and left atrial tissue). One part of the sample was used for histological analysis, including iTRAQ proteomics, immunohistochemistry, immunofluorescence and transmission electron microscope; the other part was analyzed by qRT‐PCR and western blotting to observe the molecular level of type IV collagen.ResultsiTRAQ proteomic study showed that type IV collagen was up‐regulated in right and left atrial tissue. Type IV collagen was located around cardiomyocytes, especially on vascular basement membrane and myocardial cell membrane. The fluorescence intensity of type IV collagen increased in AF patients. Electron microscope revealed that there was a large number of immature fibrocytes among myocytes, likely immature type collagen IV. qRT‐PCR and western blots showed that the mRNA and protein of type IV collagen were significantly elevated in AF patients, especially in the left atrial tissue.ConclusionsOur study identified that the change of type IV collagen in the atrial appendage is related to the development of AF in the mitral valvular disease. The present study suggests that abnormality of the type IV collagen may play an important role in the structure and electrical signal in the heart valvular disease associated with AF.Support or Funding InformationSupported by National Natural Science Foundation of China (No.81170148), Zhejiang Provincial Natural Science Foundation (LY15H020008), Hangzhou Science and Technology Projects (20140633B12), National Science and Technology Major Project (2013ZX09303004‐005), &amp; Tianjin Binhai Key Platform for Creative Research Program (2012‐BH110004), Binhai New Area Health Bureau (2012BWKZ008,2014BWKY002, 2014BWKY010), Tianjin Health Bureau (2013KZ009,2014KZ005).

Cochlear changes in serous labyrinthitis associated with silent otitis media: A human temporal bone study

PurposeTo determine histopathological findings in the cochlea of human temporal bones with serous labyrinthitis. Materials and methodsWe compared human temporal bones with serous labyrinthitis (20 cases) associated with silent otitis media and without serous labyrinthitis (20 cases) to study location of serous labyrinthitis, the degree of endolymphatic hydrops, number of spiral ganglion cells and hair cells, loss of fibrocytes in the spiral ligament, and areas of the spiral ligament and stria vascularis. ResultsThe serous labyrinthitis caused significant loss of outer hair cells in the lower basal (P=0.006), upper basal (P=0.005), and lower middle (P=0.011) cochlear turns, and significant increase in the degree of endolymphatic hydrops than the control group (P=0.036). No significant difference was found in the loss of inner hair cells, in the number of spiral ganglion cells and fibrocytes in the spiral ligament, and in areas of the stria vascularis and spiral ligament (P>0.05). ConclusionsSerous labyrinthitis resulted in significant loss of outer hair cells and significant increase in the degree of endolymphatic hydrops.

Involvement of fibrocytes in asthma and clinical implications.

Bloodborne fibrocytes are bone marrow-derived cells that participate in immune responses and exhibit pro-inflammatory and matrix remodelling properties. In patients with asthma receiving an adequate treatment, the blood fibrocyte count is very low and comparable to that obtained in healthy individuals. In these patients, a transient increase in fibrocyte numbers in the peripheral blood and in the airways occurs in concomitance with increased bronchial inflammation and reflects disease worsening and the need for more intensive treatment. Persistently elevated numbers of fibrocytes in the peripheral blood and in the bronchial mucosa are observed in chronically undertreated or corticosteroid-resistant asthma and are associated with persistent airway inflammation and ongoing remodelling of the bronchial wall. The asthmatic bronchial epithelium is the main source of fibrocyte chemoattractants in asthma and contributes with T helper type 2 lymphocytes and eosinophils to promote the proliferation and pro-remodelling function of recruited fibrocytes. The presence of elevated numbers of fibrocytes in the bronchial mucosa of allergic patients with undertreated or treatment-resistant asthma may also increase the risk of acute exacerbations because these cells can amplify T helper type 2 lymphocyte-driven inflammation on every exposure to the clinically relevant allergen and can promote further inflammation on rhinovirus infections by allowing viral replication and releasing additional pro-inflammatory factors. Improved methods for the isolation and functional analysis of pure populations of viable circulating fibrocytes have allowed a better understanding of the effector role of these cells. A reliable and clinically applicable assay has been developed to measure blood fibrocyte counts as outcome measure in future clinical trials. New therapeutic agents are needed to block both persistent inflammation and fibrocytosis in corticosteroid-resistant asthma.

Significance of circulating fibrocytes in early-stage renal fibrosis of renal failure rats

Objective To investigate the relationship between the number of peripheral blood fibrocytes and the degree of renal fibrosis, and explore the feasibility of peripheral blood fibrocytes as the early biomarker and prognostic indicator of renal fibrosis. Methods CD45 and type I collagen were taken as the markers of circulating fibrocytes. The flow cytometer was used to count bibrocytes in rat peripheral blood with 5/6 kidney cut and carry out identification and positioning on the infiltration of circulating fibrocytes in the kidney of rat with renal failure with immunohistochemistry, and set forth the significance of circulating fibrocyte counting in renal fibrosis combined with biochemical markers of renal function. Results For the model group of rat with renal failure, in the forth, eighth, and twelfth weeks after modeling, the peripheral blood fibrocyte counting was significantly increased (P<0.01). In the second, forth, and twelfth weeks after modeling, the blood cell counting was significantly positively correlated with renal pathology changes (r=0.613, P<0.01), and also was significantly positively correlated with serum creatinine (Scr), and blood urea nitrogen (BUN) (r=0.623, 0.550, P<0.01). Conclusions The peripheral blood fibrocyte counting was positively correlated with renal pathology changes in early renal fibrosis and might early indicate the developmental process of renal fibrosis. Key words: Fibroblasts/CY/ME; Fibrosis/ME; Kidney failure/ME

An Appreciation for the Rabbit Ladderlike Modeling of Radiation-induced Lung Injury with High-energy X-Ray.

Background:To evaluate the utility of rabbit ladderlike model of radiation-induced lung injury (RILI) for the future investigation of computed tomography perfusion.Methods:A total of 72 New Zealand rabbits were randomly divided into two groups: 36 rabbits in the test group were administered 25 Gy of single fractionated radiation to the whole lung of unilateral lung; 36 rabbits in the control group were sham-radiated. All rabbits were subsequently sacrificed at 1, 6, 12, 24, 48, 72 h, and 1, 2, 4, 8,1 6, 24 weeks after radiation, and then six specimens were extracted from the upper, middle and lower fields of the bilateral lungs. The pathological changes in these specimens were observed with light and electron microscopy; the expression of tumor necrosis factor-α (TNF-a) and transforming growth factor-β1 (TGF-β1) in local lung tissue was detected by immunohistochemistry.Results:(1) Radiation-induced lung injury occurred in all rabbits in the test group. (2) Expression of TNF-a and TGF-β1 at 1 h and 48 h after radiation, demonstrated a statistically significant difference between the test and control groups (each P < 0.05). (3) Evaluation by light microscopy demonstrated statistically significant differences between the two groups in the following parameters (each P < 0.05): thickness of alveolar wall, density of pulmonary interstitium area (1 h after radiation), number of fibroblasts and fibrocytes in interstitium (24 h after radiation). The test group metrics also correlated well with the time of postradiation. (4) Evaluation by electron microscopy demonstrated statistically significant differences in the relative amounts of collagen fibers at various time points postradiation in the test group (P < 0.005), with no significant differences in the control group (P > 0.05). At greater than 48 h postradiation the relative amount of collagen fibers in the test groups significantly differ from the control groups (each P < 0.05), correlating well with the time postradiation (r = 0.99318).Conclusions:A consistent and reliable rabbit model of RILI can be generated in gradient using 25 Gy of high-energy X-ray, which can simulate the development and evolution of RILI.

Bone marrow-derived fibrocytes contribute to liver fibrosis

Chronic liver injury often leads to hepatic fibrosis, a condition associated with increased levels of circulating TGF-β1 and lipopolysaccharide, activation of myofibroblasts, and extensive deposition of extracellular matrix, mostly collagen Type I. Hepatic stellate cells are considered to be the major(1) but not the only source of myofibroblasts in the injured liver.(2) Hepatic myofibroblasts may also originate from portal fibroblasts, mesenchymal cells, and fibrocytes.(3) Since the discovery of fibrocytes in 1994 by Dr. Bucala and colleagues, this bone marrow (BM)-derived collagen Type I-producing CD45(+) cells remain the most fascinating cells of the hematopoietic system. Due to the ability to differentiate into collagen Type I producing cells/myofibroblasts, fibrocytes were implicated in the pathogenesis of liver, skin, lung, and kidney fibrosis. However, studies of different organs often contain controversial results on the number of fibrocytes recruited to the site of injury and their biological function. Furthermore, fibrocytes were implicated in the pathogenesis of sepsis and were shown to possess antimicrobial activity. Finally, in response to specific stimuli, fibrocytes can give rise to fully differentiated macrophages, suggesting that in concurrence with the high plasticity of hematopoietic cells, fibrocytes exhibit progenitor properties. Here, we summarize our current understanding of the role of CD45(+)Collagen Type I(+) BM-derived cells in response to fibrogenic liver injury and septicemia and discuss the most recent evidence supporting the critical role of fibrocytes in the mediation of pro-fibrogenic and/or pro-inflammatory responses.

Kruppel-Like Factor KLF4 Facilitates Cutaneous Wound Healing by Promoting Fibrocyte Generation from Myeloid-Derived Suppressor Cells

Pressure ulcers (PUs) are serious skin injuries whereby the wound healing process is frequently stalled in the inflammatory phase. Myeloid-derived suppressor cells (MDSCs) accumulate as a result of inflammation and promote cutaneous wound healing by mechanisms not fully understood. Recently, MDSCs have been shown to differentiate into fibrocytes which serve as emerging effector cells that enhance cell proliferation in wound healing. We postulate that in wound healing, MDSCs not only execute their immunosuppressive function to regulate inflammation, but also stimulate cell proliferation once they differentiate into fibrocytes. In the current study, using full thickness and pressure ulcer mouse models, we found that KLF4 deficiency resulted in decreased accumulation of MDSCs and fibrocytes and wound healing was significantly delayed. Conversely, KLF4 activation by the plant-derived product, Mexicanin I, increased the numbers of MDSCs and fibrocytes and accelerated wound healing. Collectively, our study revealed a previously unreported function of MDSCs in cutaneous wound healing and identified Mexicanin I as a potential agent to accelerate PU wound healing.

Contribution of bone marrow-derived fibrocytes to liver fibrosis.

Since the discovery of fibrocytes in 1994 by Dr. Bucala and colleagues, these bone marrow (BM)-derived collagen Type I producing CD45(+) cells remain the most fascinating cells of the hematopoietic system. Despite recent reports on the emerging contribution of fibrocytes to fibrosis of parenchymal and non-parenchymal organs and tissues, fibrocytes remain the most understudied pro-fibrogenic cellular population. In the past years fibrocytes were implicated in the pathogenesis of liver, skin, lung, and kidney fibrosis by giving rise to collagen type I producing cells/myofibroblasts. Hence, the role of fibrocytes in fibrosis is not well defined since different studies often contain controversial results on the number of fibrocytes recruited to the site of injury versus the number of fibrocyte-derived myofibroblasts in the same fibrotic organ. Furthermore, many studies were based on the in vitro characterization of fibrocytes formed after outgrowth of BM and/or peripheral blood cultures. Therefore, the fibrocyte function(s) still remain(s) lack of understanding, mostly due to (I) the lack of mouse models that can provide complimentary in vivo real-time and cell fate mapping studies of the dynamic differentiation of fibrocytes and their progeny into collagen type I producing cells (and/or possibly, other cell types of the hematopoietic system); (II) the complexity of hematopoietic cell differentiation pathways in response to various stimuli; (III) the high plasticity of hematopoietic cells. Here we summarize the current understanding of the role of CD45(+) collagen type I(+) BM-derived cells in the pathogenesis of liver injury. Based on data obtained from various organs undergoing fibrogenesis or other type of chronic injury, here we also discuss the most recent evidence supporting the critical role of fibrocytes in the mediation of pro-fibrogenic and/or pro-inflammatory responses.

The Contribution of Fibrocytes to the Progression of Multiple Myeloma

Background: Fibrocytes are circulating, pro-collagen expressing myeloid cells that contribute to wound healing. Emerging data implicates these cells in cancer progression. Our data supports a role for fibrocytes in the progression of multiple myeloma (MM). Methods: Peripheral blood and bone marrow samples were collected according to an IRB approved protocol (IRB Number: 09-0768). Fibrocytes were identified in the PBMC fraction by flow cytometry as CD45+, COL1A1+, and CD115-cells. These results were confirmed by counting fibrocytes after five days of culture in serum-free conditions. Statistical analysis was performed by dividing the sample set into four subgroups: Monoclonal Gammopathy of Unknown Significance (MGUS), Smoldering Myeloma (SM), treated myeloma (TrMM), and untreated or relapsed myeloma (U/RMM). Significance was determined using non-parametric methods with the Bonferroni correction for multiple group comparisons. The effects of MM cells on fibrocyte number were studied in vitro using a transwell system that co-cultured healthy donor PBMCs with MM cells from U/RMM patients. MM samples from patients with a greater than average number of circulating fibrocytes were compared with samples from patients with fewer than average. Results were reported as the fold increase in fibrocyte number compared with donor PBMCs alone. Differences in gene expression between fibrocytes from five U/RMM and five MGUS patients were assessed using a RT2Profiler PCR Custom Array (Qiagen). Fibrocytes were isolated by culturing PBMCs in serum free conditions followed by immunomagnetic bead purification. Results: Eighty-eight patient samples were analyzed. There were no differences in age, WBC count, monocyte count, hemoglobin, albumin, or creatinine. Measures of disease burden including β2M, percentage of bone marrow plasma cells, and concentration of the M-protein were consistent with the patients’ strata. U/RMM patients had an increased number of circulating fibrocytes (4.7 ± 0.6 x107 cells/L) compared with TrMM (2.8 ± 0.5 x107 cells/L), SM (2.0 ± 0.5 x107 cells/L), MGUS patients (2.0 ± 0.6 x107 cells/L), or healthy controls (0.8 ± 0.2 x107 cells/L) (p &lt; 0.05). These differences were confirmed using the cell culture techniques. Twenty-nine patients with MGUS or SM were not treated when entering the study. Seven of these developed progressive disease that required therapy. This group had a significantly greater number of circulating fibrocytes compared with the 22 patients without progression (2.4 ± 0.7 vs 1.3 ± 0.4 x 107 cells/L, p&lt;0.05). Primary MM cells were isolated from U/RMM patients with greater than average circulating fibrocytes (6.5 ± 1.2 x 107 cells/L) and those with less than average fibrocytes (1.8 ± 0.6 x 107 cells/L). The co-culture of these cells with healthy donor PBMCs increased the number of fibrocytes by 3.9 ± 0.9 fold. This increase was significantly greater using MM cells from patients with more circulating fibrocytes compared with those with less (5.4 ± 1.5 vs 2.5 ± 0.3 fold; p&lt;0.05). Twenty-eight potential mediators of this process were compiled from the literature and were assessed using bioinformatics applied to public databases. Of these, SPARC was the most promising candidate based on its differential expression in MM samples compared with normal plasma cells. Real time PCR measurement of SPARC expression in our U/RMM samples was positively correlated with the number of circulating fibrocytes (r=0.982, p=0.018). We began to explore the ability of fibrocytes to promote MM by screening these cells using a custom real time PCR array. Twenty genes were selected for analysis based on our work using murine models. Of these, SPP1 was expressed at least one log greater than any of the other 19 genes (p &lt; 0.01). Furthermore, fibrocytes from U/RMM patients had significantly greater expression of SPP1 than MGUS patients (1.82 fold, p&lt;0.05). Proposed Model: Our data support a model in which MM cells produce SPARC. This signal causes an expansion of fibrocytes. These cells, in turn, promote MM progression by expressing SPP1, a gene that can activate osteoclasts leading to a release of MM growth factors. In this way, fibrocytes can participate in a positive feedback loop leading to progression of myeloma. Disclosures No relevant conflicts of interest to declare.

CCR1-Mediated Formation of Liver Microenvironment for Cancer Metastasis

Accumulating evidence suggests that bone marrow-derived myeloid cells constitute the major components of cancer microenvironment and play significant roles in the progression of cancer. We have previously shown that myeloid cells expressing CCR1, a chemokine receptor, are recruited to the microenvironment of disseminated colon cancer cells in the liver (PNAS 2010). These CCR1+ cells promote metastatic colonization of cancer cells in the liver through producing metalloproteinases MMP9 and MMP2. However, the cell lineage and precise characteristics of these myeloid cells were largely unknown. The aim of this study is to characterize the cell types among these myeloid cells expressing CCR1 and/or MMPs.In order to characterize the CCR1-expressing myeloid cells accumulating at liver metastatic foci, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1cis-regulatory elements. A flow cytometric analysis of adult transgenic mouse bone marrow cells showed that expression of the reporter mVenus fluorescence was restricted to the CD11b+ Gr-1+ cells and the level of endogenous Ccr1 mRNA correlated well with that of mVenus fluorescence, suggesting that expression of the reporter precisely reflected that of the endogenous CCR1.Inoculation of CCL9-expressing mouse colon cancer cell line CMT93 into the spleen of syngeneic hosts causes efficient metastatic colonization of the cancer cells in the liver. Employing this model, we monitored the behavior of the Ccr1-mVenus+ myeloid cells in the cancer metastasis microenvironment, and found that CD45+ CD11b+Ccr1-mVenus+ cells purified from liver metastatic foci were mostly neutrophils. In contrast, within CD45+ CD11b+Ccr1-mVenus– fraction, we identified eosinophils, monocytes and fibrocytes. Notably, the major cell types in the metastatic foci were monocytes and fibrocytes, with the latter producing abundant collagen.Chronologically, accumulation of neutrophils and monocytes preceded that of eosinophils and fibrocytes. In Ccr1−/− mice, early accumulation of Ccr1-mVenus+ Gr-1+ cells was significantly compromised and later recruitment of fibrocytes to the metastatic foci was reduced. Antibody-mediated depletion of neutrophils caused a significant reduction in the fibrocyte number at the metastatic foci. These results suggest that CCR1 plays critical roles in early accumulation of neutrophils and subsequent recruitment of fibrocytes.After having identified four types of myeloid cells in the metastatic foci, we determined the expression levels of CCR1, MMP9 and MMP2 mRNAs in each purified population, and found that CCR1 and MMP9 are expressed preferentially by neutrophils, whereas MMP2 is expressed exclusively by monocytes/fibrocytes. Considering the chronological difference in the appearance of myeloid cells in liver metastatic foci, these results suggest that production of MMP9 and MMP2 in succession by different myeloid cells is necessary for disseminated colon cancer cells to colonize in the liver.In summary, these results suggests that CCR1 is required for recruitment of neutrophils in the early phase of colon cancer dissemination and the subsequent accumulation of fibrocytes, and that MMP9 and MMP2 are produced by neutrophils and fibrocytes, respectively, in a successive manner during the formation of liver metastasis. Further investigation of these cancer-driven host reactions in human colon cancer dissemination may lead to effective preventive measures against cancer metastasis. DisclosuresNo relevant conflicts of interest to declare.

Role of platelet-derived growth factor/platelet-derived growth factor receptor axis in the trafficking of circulating fibrocytes in pulmonary fibrosis.

Circulating fibrocytes have been reported to migrate into the injured lungs, and contribute to fibrogenesis via CXCL12-CXCR4 axis. In contrast, we report that imatinib mesylate prevented bleomycin (BLM)-induced pulmonary fibrosis in mice by inhibiting platelet-derived growth factor receptor (PDGFR), even when it was administered only in the early phase. The goal of this study was to test the hypothesis that platelet-derived growth factor (PDGF) might directly contribute to the migration of fibrocytes to the injured lungs. PDGFR expression in fibrocytes was examined by flow cytometry and RT-PCR. The migration of fibrocytes was evaluated by using a chemotaxis assay for human fibrocytes isolated from peripheral blood. The numbers of fibrocytes triple-stained for CD45, collagen-1, and CXCR4 were also examined in lung digests of BLM-treated mice. PDGFR mRNA levels in fibrocytes isolated from patients with idiopathic pulmonary fibrosis were investigated by real-time PCR. Fibrocytes expressed both PDGFR-α and -β, and migrated in response to PDGFs. PDGFR inhibitors (imatinib, PDGFR-blocking antibodies) suppressed fibrocyte migration in vitro, and reduced the number of fibrocytes in the lungs of BLM-treated mice. PDGF-BB was a stronger chemoattractant than the other PDGFs in vitro, and anti-PDGFR-β-blocking antibody decreased the numbers of fibrocytes in the lungs compared with anti-PDGFR-α antibody in vivo. Marked expression of PDGFR-β was observed in fibrocytes from patients with idiopathic pulmonary fibrosis compared with healthy subjects. These results suggest that PDGF directly functions as a strong chemoattractant for fibrocytes. In particular, the PDGF-BB-PDGFR-β biological axis might play a critical role in fibrocyte migration into the fibrotic lungs.

Fibrocytes in the fibrotic lung: altered phenotype detected by flow cytometry.

Fibrocytes are bone marrow hematopoietic-derived cells that also express a mesenchymal cell marker (commonly collagen I) and participate in fibrotic diseases of multiple organs. Given their origin, they or their precursors must be circulating cells before recruitment into target tissues. While most previous studies focused on circulating fibrocytes, here we focus on the fibrocyte phenotype in fibrotic tissue. The study's relevance to human disease is heightened by use of a model in which bleomycin is delivered systemically, recapitulating several features of human scleroderma including multi-organ fibrosis not observed when bleomycin is delivered directly into the lungs. Using flow cytometry, we find in the fibrotic lung a large population of CD45high fibrocytes (called Region I) rarely found in vehicle-treated control mice. A second population of CD45+ fibrocytes (called Region II) is observed in both control and fibrotic lung. The level of CD45 in circulating fibrocytes is far lower than in either Region I or II lung fibrocytes. The chemokine receptors CXCR4 and CCR5 are expressed at higher levels in Region I than in Region II and are present at very low levels in all other lung cells including CD45+/collagen I- leucocytes. The collagen chaperone HSP47 is present at similar high levels in both Regions I and II, but at a higher level in fibrotic lung than in control lung. There is also a major population of HSP47high/CD45- cells in fibrotic lung not present in control lung. CD44 is present at higher levels in Region I than in Region II and at much lower levels in all other cells including CD45+/collagen I- leucocytes. When lung fibrosis is inhibited by restoring caveolin-1 activity using a caveolin-1 scaffolding domain peptide (CSD), a strong correlation is observed between fibrocyte number and fibrosis score. In summary, the distinctive phenotype of fibrotic lung fibrocytes suggests that fibrocyte differentiation occurs primarily within the target organ.

304. Modulating the Fibrotic Effects of Fibrocytes from Scleroderma Patients

Background: SSc is a complex autoimmune fibrotic disease, characterized by elevated deposition of extracellular matrix (ECM) proteins, in particular collagen type I. The disease is heterogeneous; organs commonly affected by fibrosis are the skin, kidney, lung and heart. Vascular complications include pulmonary arterial hypertension (PAH), which occurs in 12–40% of patients. CD14þmonocytes are afunctionally heterogeneous cell type. They have been noted to differentiate into a number of cell phenotypes including macrophages and collagen-producing fibrocytes. In culture fibrocytes adopt a spindle shape, co-express haematopoietic CD45RO and 25F9, along with mesenchymal markers including aSMA and collagen type I. Fibrocytes amplify the inflammatory/immune response through distinct mechanisms, including antigen presentation, cytokine and chemokine secretion, and the production of MMPs. We and others have shown fibrocyte differentiation is enhanced by fibrogenic cytokines including PDGF. Here we seek to understand the mechanism by which SSc fibrocytes influence the local microenvironment of the tissue. Methods: CD14þ peripheral blood mononuclear cells (PBMCs) were isolated from SSc patient and healthy control blood. PBMCs were cultured in the presence of macrophage colony stimulating factor (MCSF; n > 10) and/or endothelin-1 (ET-1; n > 10); after 14 days of culture number of fibrocytes was assessed. The effect of pharmacological inhibitors including ETRA and ETRB antagonism on fibrocyte differentiation (n ¼ 6 SSc and control) was investigated. Secreted factors in culture media from SSc and control fibrocytes were assessed by ELISA (n ¼ 6), and the effects of conditioned media explored in 3D-collagen gel. Results: Both MCSF and ET-1 significantly induced fibrocyte differentiation, in combination differentiation was significantly augmented (P < 0.05) in comparison with mono-treatment. SSc fibrocytes more readily differentiated from CD14þ PBMCs than healthy control donors in response to MCSF (P < 0.05), ET-1 (P < 0.05) as well as MCSF with ET-1 in combination (P < 0.01). ETRA and ETRB antagonists, BQ123 and BQ788 (respectively), and Bosentan (a dual ETR antagonist) inhibited MCSF induced fibrocyte differentiation in a concentration dependant manner. Furthermore SSc fibrocytes secreted significantly more connective tissue growth factor (CTGF) than control fibrocytes (P < 0.05) cultured with MCSF. Consistent with fibrocytes acting in a paracrine manner, conditioned media from SSc fibrocytes promoted fibroblast gel contraction by control cells (P < 0.05). Conclusion: Here we show CD14þ SSc PBMCs more readily differentiate into fibrocytes and that activation via the ETRA and ETRB is essential for ET-1 and MCSF induced fibrocyte differentiation. Suggesting MCSF acts indirectly via ET-1 release; possibly resulting in a positive feedback loop. Our data suggest fibrocytes may contribute to the development of a pro-fibrotic environment through influencing tissue resident fibroblasts in a paracrine manner

Possible Involvement of Fibrocytes in Atrial Fibrosis in Patients With Chronic Atrial Fibrillation

Chronic atrial fibrillation (AF) is characterized by a remodeling process with prominent atrial fibrosis. Fibrocytes, a bone marrow-derived population of fibroblast-like cells, have been placed at the center of a number of fibrosing conditions. The purpose of this study was to evaluate the contribution of fibrocytes to atrial fibrosis in patients with chronic AF and the possible mechanisms. We enrolled 22 consecutive valvular heart disease patients with chronic AF (>6 months: CAF group) and 15 valvular heart disease patients in sinus rhythm served as controls (SR group). Left atrial tissue samples were obtained during cardiac surgery. The infiltration of fibrocytes into the atrial interstitium was observed by confocal microscopy. The number of atrial fibrocytes was approximately three-fold higher in the CAF group compared with the SR controls, and positively correlated with both the atrial collagen volume fraction (r=0.713; P=0.0002) and the left atrial volume index (r=0.631; P=0.002). In the peripheral blood samples collected before the operation, approximately 2.5-fold higher percentage of circulating fibrocytes was identified in the CAF group. These fibrocytes showed a stronger proliferative capacity (≍2.5-fold) and higher level expression of collagen I and α-SMA (≍2-fold and 4-fold, respectively) compared with the SR controls. The results suggested that fibrocytes may be involved in atrial fibrosis in chronic AF through enhanced profibrotic characteristics.

Histological Analysis of Collagen Fibers in Patients with Diabetes Mellitus and Periodontal Disease

The researchers suggest that diabetes increases the degradation of the newly synthesized collagen from the conjunctive tissue. The altered metabolism of the collagen may predispose the diabetic patients not only to periodontal disease, but also to some other anomalies such as defective healing. The purpose of the study was to go through a histological analysis of the collagen fibers from the chorion in patients with diabetes mellitus and periodontal disease. Materials and Methods: In our study we examined a total of 68 patients diagnosed with diabetes mellitus type 1 and 2. In these patients we achieved simple gingivectomy or tooth extraction in order to obtain gingival tissue fragments. The preparations included at paraffin and then stained with Hematoxylin-Eosin and trichromic after Goldner-Szeckeli method. Results: In the patients with diabetes mellitus with an evolution of less than 10 years the collagen fibers appeared as dissociated by the inflammatory cells that were inserted among them. It was also noted the fragmentation of collagen fibers and sometimes even their lysis with subepithelial connective tissue disorganization. In patients with diabetes for over 10 years there has been noticed a plentiful collagen sclerosis, zones of collagen hyalinization with an increased number of fibrocytes. Conclusion: The extracellular matrix is altered in the investigated patients with destructive lesions of collagen or collagen sclerosis.

New Light Is Shed on the Enigmatic Origin of the Lung Myofibroblast

New Light Is Shed on the Enigmatic Origin of the Lung Myofibroblast

Extracorporeal cardiac shock wave therapy ameliorates myocardial fibrosis by decreasing the amount of fibrocytes after acute myocardial infarction in pigs

Extracorporeal shock wave (SW) therapy ameliorates cardiac remodeling after acute myocardial infarction (AMI). However, it remains to be examined whether and how SW therapy ameliorates myocardial fibrosis after AMI. Fibrocytes are associated with myocardial fibrosis. Thus, we examined whether SW therapy ameliorates myocardial fibrosis and whether fibrocytes are associated after AMI in pigs. AMI was created by coronary embolism. Twenty-five pigs were divided into three groups: AMI+SW group (AMI with SW therapy, n=15), AMI group (without SW therapy, n=5), and sham+SW group (SW therapy without AMI, n=5). The collagen area fraction was examined by Masson's trichrome staining. The presence of fibrocytes was identified by immunofluorescence and confocal microscopy. The location of CXCL12 was examined by immunohistochemistry. Compared with the AMI group, the AMI+SW group showed significantly ameliorated myocardial fibrosis in terms of collagen area fraction (27.21±8.13 vs. 10.13±4.96, P<0.05) and reduced fibrocytes (CD34/α-smooth muscle actin: 35.40±11.72 vs. 12.27±7.71, P<0.05; CXCR4/α-smooth muscle actin: 40.80±8.96 vs. 16.54±6.38, P<0.05). There were positive correlations between the collagen area fraction and the number of fibrocytes (r=0.936; P<0.05) and between the number of CXCR4 fibrocytes and the SDF-1/CXCL12 cells (r=0.802; P<0.05) in the three groups. The results show that SW therapy ameliorates myocardial fibrosis after AMI in pigs, which is associated with the decreased amount of fibrocytes.