Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen

Background

Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D) environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP) expression in rat vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D.

Methodology/Principal Findings

Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS) chains from proteoglycan (PG) core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1) suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13) expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK) also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs) were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity.

Conclusions/Significance

We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D. This is the first study to describe a flow-induced mechanotransduction mechanism via HSPG-mediated FAK activation in 3D. This study will be of interest in understanding the flow-related mechanobiology in vascular lesion formation, tissue morphogenesis, cancer cell metastasis, and stem cell differentiation in 3D, and also has implications in tissue engineering.     

Delayed re-epithelialization in periostin-deficient mice during cutaneous wound healing

Background

Matricellular proteins, including periostin, are important for tissue regeneration.

Methods and Findings

Presently we investigated the function of periostin in cutaneous wound healing by using periostin-deficient (−/−) mice. Periostin mRNA was expressed in both the epidermis and hair follicles, and periostin protein was located at the basement membrane in the hair follicles together with fibronectin and laminin γ2. Periostin was associated with laminin γ2, and this association enhanced the proteolytic cleavage of the laminin γ2 long form to produce its short form. To address the role of periostin in wound healing, we employed a wound healing model using WT and periostin−/− mice and the scratch wound assay in vitro. We found that the wound closure was delayed in the periostin−/− mice coupled with a delay in re-epithelialization and with reduced proliferation of keratinocytes. Furthermore, keratinocyte proliferation was enhanced in periostin-overexpressing HaCaT cells along with up-regulation of phosphorylated NF-κB.

Conclusion

These results indicate that periostin was essential for keratinocyte proliferation for re-epithelialization during cutaneous wound healing.     

Extracellular and Intracellular Mechanisms of Mechanotransduction in Three-Dimensionally Embedded Rat Chondrocytes

Purpose

Articular cartilage homeostasis involves modulation of chondrocyte matrix synthesis in response to mechanical stress (MS). We studied extracellular and intracellular mechanotransduction pathways mediating this response.

Methods

We first confirmed rapid up-regulation of the putative chondro-protective cytokine, interleukin (IL)-4, as an immediate response to MS. We then studied the role of IL-4 by investigating responses to exogenous IL-4 or a specific IL-4 inhibitor, combined with MS. Next we investigated the intracellular second messengers. Since chondrocyte phenotype alters according to the extracellular environment, we characterized the response to mechanotransduction in 3-dimensionally embedded chondrocytes.

Results

Expression of aggrecan and type II collagen was significantly up-regulated by exogenous IL-4 whereas MS-induced matrix synthesis was inhibited by an IL-4 blocker. Further, MS-induced matrix synthesis was completely blocked by a p38 MAPK inhibitor, while it was only partially blocked by inhibitors of other putative second messengers.

Conclusion

IL-4 mediates an extracellular pathway of mechanotransduction, perhaps via an autocrine/paracrine loop, while p38 mediates an intracellular pathway prevalent only in a 3-dimensional environment.     

Inactivation of Fam20C in Cells Expressing Type I Collagen Causes Periodontal Disease in Mice

Background

FAM20C is a kinase that phosphorylates secretory proteins. Previous studies have shown that FAM20C plays an essential role in the formation and mineralization of bone, dentin and enamel. The present study analyzed the loss-of-function effects of FAM20C on the health of mouse periodontal tissues.

Methods

By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20C^fl/fl mice, we created 2.3 kb Col 1a1-Cre;Fam20C^fl/fl (cKO) mice, in which Fam20C was inactivated in the cells that express Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology, scanning electron microscopy and immunohistochemistry approaches.

Results

The cKO mice underwent a remarkable loss of alveolar bone and cementum, along with inflammation of the periodontal ligament and formation of periodontal pockets. The osteocytes and lacuno-canalicular networks in the alveolar bone of the cKO mice showed dramatic abnormalities. The levels of bone sialoprotein, osteopontin, dentin matrix protein 1 and dentin sialoprotein were reduced in the Fam20C-deficient alveolar bone and/or cementum, while periostin and fibrillin-1 were decreased in the periodontal ligament of the cKO mice.

Conclusion

Loss of Fam20C function leads to periodontal disease in mice. The reduced levels of bone sialoprotein, osteopontin, dentin matrix protein 1, dentin sialoprotein, periostin and fibrillin-1 may contribute to the periodontal defects in the Fam20C-deficient mice.     

Periostin Associates with Notch1 Precursor to Maintain Notch1 Expression under a Stress Condition in Mouse Cells

Background

Matricellular proteins, including periostin, modulate cell-matrix interactions and cell functions by acting outside of cells.

Methods and Findings

In this study, however, we reported that periostin physically associates with the Notch1 precursor at its EGF repeats in the inside of cells. Moreover, by using the periodontal ligament of molar from periostin-deficient adult mice (Pn−/− molar PDL), which is a constitutively mechanically stressed tissue, we found that periostin maintained the site-1 cleaved 120-kDa transmembrane domain of Notch1 (N1™) level without regulating Notch1 mRNA expression. N1™ maintenance in vitro was also observed under such a stress condition as heat and H₂O₂ treatment in periostin overexpressed cells. Furthermore, we found that the expression of a downstream effector of Notch signaling, Bcl-xL was decreased in the Pn−/− molar PDL, and in the molar movement, cell death was enhanced in the pressure side of Pn−/− molar PDL.

Conclusion

These results suggest the possibility that periostin inhibits cell death through up-regulation of Bcl-xL expression by maintaining the Notch1 protein level under the stress condition, which is caused by its physical association with the Notch1 precursor.     

Associations between the Duration of Dialysis,Endotoxemia, Monocyte Chemoattractant Protein-1, and the Effects of a Short-Dwell Exchange in Patients Requiring Continuous Ambulatory Peritoneal Dialysis

Background

Endotoxemia is exaggerated and contributes to systemic inflammation and atherosclerosis in patients requiring continuous ambulatory peritoneal dialysis (CAPD). The risk of mortality is substantially increased in patients requiring CAPD for >2 years. However, little is known about the effects of long-term CAPD on circulating endotoxin and cytokine levels. Therefore, the present study evaluated the associations between plasma endotoxin levels, cytokine levels, and clinical parameters with the effects of a short-dwell exchange on endotoxemia and cytokine levels in patients on long-term CAPD.

Methods

A total of 26 patients were enrolled and divided into two groups (short-term or long-term CAPD) according to the 2-year duration of CAPD. Plasma endotoxin and cytokine levels were measured before and after a short-dwell exchange (4-h dwell) during a peritoneal equilibration test (a standardized method to evaluate the solute transport function of peritoneal membrane). These data were analyzed to determine the relationship of circulating endotoxemia, cytokines and clinical characteristics between the two groups.

Results

Plasma endotoxin and monocyte chemotactic protein-1 (MCP-1) levels were significantly elevated in the long-term group. PD duration was significantly correlated with plasma endotoxin (r = 0.479, P = 0.016) and MCP-1 (r = 0.486, P = 0.012). PD duration was also independently associated with plasma MCP-1 levels in multivariate regression. Plasma MCP-1 levels tended to decrease (13.3% reduction, P = 0.077) though endotoxin levels did not decrease in the long-term PD group after the 4-h short-dwell exchange.

Conclusion

Long-term PD may result in exaggerated endotoxemia and elevated plasma MCP-1 levels. The duration of PD was significantly correlated with circulating endotoxin and MCP-1 levels, and was an independent predictor of plasma MCP-1 levels. Short-dwell exchange seemed to have favorable effects on circulating MCP-1 levels in patients on long-term PD.     

ACE Inhibitors Potently Reduce Vascular Inflammation,Results of an Open Proof-Of-Concept Study in the Abdominal Aortic Aneurysm

Background

Independent of their blood pressure lowering effect, ACE inhibitors are thought to reduce vascular inflammation. The clinical relevance of this effect is unclear with the current knowledge. Abdominal aortic aneurysms (AAA) are characterized by a broad, non-specific inflammatory response, and thus provide a clinical platform to evaluate the anti-inflammatory potential of ACE inhibitors.

Methods and Results

Eleven patients scheduled for open AAA repair received ramipril (5 mg/day) during 2–4 weeks preceding surgery. Aortic wall samples were collected during surgery, and compared to matched samples obtained from a biobank. An anti-inflammatory potential was evaluated in a comprehensive analysis that included immunohistochemistry, mRNA and protein analysis. A putative effect of ACE inhibitors on AAA growth was tested separately by comparing 18-month growth rate of patients on ACE inhibitors (n = 82) and those not taking ACE inhibitors (n = 204). Ramipril reduces mRNA expression of multiple pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, TNF -α, Interferon-, and MCP-1, as well as aortic wall IL-8 and MCP-1 (P = 0.017 and 0.008, respectively) protein content. The is followed by clear effects on cell activation that included a shift towards anti-inflammatory macrophage (M2) subtype. Evaluation of data from the PHAST cohort did not indicate an effect of ACE inhibitors on 18-month aneurysm progression (mean difference at 18 months: −0.24 mm (95% CI: −0.90–0.45, P = NS).

Conclusions

ACE inhibition quenches multiple aspects of vascular inflammation in AAA. However, this does not translate into reduced aneurysm growth.

Trial Registration

Nederlands Trial Register 1345.     

Mechanical Analysis of Single Myocyte Contraction in a 3-D Elastic Matrix

Background

Cardiac myocytes experience mechanical stress during each heartbeat. Excessive mechanical stresses under pathological conditions cause functional and structural remodeling that lead to heart diseases, yet the precise mechanisms are still incompletely understood. To study the cellular and molecular level mechanotransduction mechanisms, we developed a new ‘cell-in-gel’ experimental system to exert multiaxial (3-D) stresses on a single myocyte during active contraction.

Methods

Isolated myocytes are embedded in an elastic hydrogel to simulate the mechanical environment in myocardium (afterload). When electrically stimulated, the in-gel myocyte contracts while the matrix resists shortening and broadening of the cell, exerting normal and shear stresses on the cell. Here we provide a mechanical analysis, based on the Eshelby inclusion problem, of the 3-D strain and stress inside and outside the single myocyte during contraction in an elastic matrix.

Results

(1) The fractional shortening of the myocyte depends on the cell’s geometric dimensions and the relative stiffness of the cell to the gel. A slender or softer cell has less fractional shortening. A myocyte of typical dimensions embedded in a gel of similar elastic stiffness can contract only 20% of its load-free value. (2) The longitudinal stress inside the cell is about 15 times the transverse stress level. (3) The traction on the cell surface is highly non-uniform, with a maximum near its ends, showing ‘hot spots’ at the location of intercalated disks. (4) The mechanical energy expenditure of the myocyte increases with the matrix stiffness in a monotonic and nonlinear manner.

Conclusion

Our mechanical analyses provide analytic solutions that readily lend themselves to parametric studies. The resulting 3-D mapping of the strain and stress states serve to analyze and interpret ongoing cell-in-gel experiments, and the mathematical model provides an essential tool to decipher and quantify mechanotransduction mechanisms in cardiac myocytes.     

Porphyromonas gingivalis participates in pathogenesis of human abdominal aortic aneurysm by neutrophil activation. Proof of concept in rats

Background

Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia.

Methods and Findings

Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed.

Conclusions

Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression.     

Combined influence of dietary restriction and treadmill running on MCP-1 and the expression of oxidative stress-related mRNA in the adipose tissue in obese mice

[Purpose]

This study suggests that the negative effects of inflammation caused by obesity could be prevented through diet restriction and exercise.

[Methods]

In this study, 44 C57/BL6 male mice at about 4 weeks old (Orient bio, South Korea) were given a high fat diet for 5 weeks to make them obese. To help the mice lose weight, their dietary intake was limited and they were exercised on the treadmill for 8 weeks, and during that period, we analyzed the changes of MCP-1, ERK, Mn-SOD, HIF-1, and NOX in epididymal adipose tissue. There ND control group and obese group with high fat diet (HFD), and it is divided into four groups; HFD-ND-EX group, HFD-ND-nonEX group, HFD-DR-EX group and HFD-DR-nonEX group.

[Results]

During their progress, the mRNA expressions of HIF-1α and ERK2 decreased, as did the expression of MCP-1 contained in the nucleus by suppressing oxygen free radicals, which was observed after the exercise program. However, dietary restriction without exercise training triggered an increase in the mRNA expression of MCP-1.

[Conclusion]

To put this in perspective, combining exercise and dietary intake restriction likely prevented an influx of macrophages by reducing the number of fat cells, whereas only dietary restriction was not effective against reducing inflammation.     

In vivo imaging of macrophages during the early-stages of abdominal aortic aneurysm using high resolution MRI in ApoE mice

Background

Angiotensin II (ANG II) promotes vascular inflammation and induces abdominal aortic aneurysm (AAA) in hyperlipidemic apolipoprotein E knock-out (apoE^−/−) mice. The aim of the present study was to detect macrophage activities in an ANG II-induced early-stage AAA model using superparamagnetic iron oxide (SPIO) as a marker.

Methodology/Principal Findings

Twenty-six male apoE^−/− mice received saline or ANG II (1000 or 500 ng/kg/min) infusion for 14 days. All animals underwent MRI scanning following administration of SPIO with the exception of three mice in the 1000 ng ANG II group, which were scanned without SPIO administration. MR imaging was performed using black-blood T2 to proton density -weighted multi-spin multi-echo sequence. In vivo MRI measurement of SPIO uptake and abdominal aortic diameter were obtained. Prussian blue, CD68,α-SMC and MAC3 immunohistological stains were used for the detection of SPIO, macrophages and smooth muscle cells. ANG II infusion with 1000 ng/kg/min induced AAA in all of the apoE^−/− mice. ANG II infusion exhibited significantly higher degrees of SPIO uptake, which was detected using MRI as a distinct loss of signal intensity. The contrast-to-noise ratio value decreased in proportion to an increase in the number of iron-laden macrophages in the aneurysm. The aneurysmal vessel wall in both groups of ANG II treated mice contained more iron-positive macrophages than saline-treated mice. However, the presence of cells capable of phagocytosing haemosiderin in mural thrombi also induced low-signal-intensities via MRI imaging.

Conclusions/Significance

SPIO is taken up by macrophages in the shoulder and the outer layer of AAA. This alters the MRI signaling properties and can be used in imaging inflammation associated with AAA. It is important to compare images of the aorta before and after SPIO injection.     

Circulating Endothelial Progenitor Cell and Platelet Microparticle Impact on Platelet Activation in Hypertension Associated with Hypercholesterolemia

Aim

The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs) and platelet microparticles (PMPs) on blood platelet function in experimental hypertension associated with hypercholesterolemia.

Methods

Golden Syrian hamsters were divided in six groups: (i) control, C; (ii) hypertensive-hypercholesterolemic, HH; (iii) ‘prevention’, HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv) ‘regression’, HHfin-EPCs, HH treated with EPCs after HH feeding; (v) HH treated with PMPs, HH-PMPs, and (vi) HH treated with EPCs and PMPs, HH-EPCs-PMPs.

Results

Compared to HH group, the platelets from HHin-EPCs and HHfin-EPCs groups showed a reduction of: (i) activation, reflected by decreased integrin 3β, FAK, PI3K, src protein expression; (ii) secreted molecules as: SDF-1, MCP-1, RANTES, VEGF, PF4, PDGF and (iii) expression of pro-inflammatory molecules as: SDF-1, MCP-1, RANTES, IL-6, IL-1β; TFPI secretion was increased. Compared to HH group, platelets of HH-PMPs group showed increased activation, molecules release and proteins expression. Compared to HH-PMPs group the combination EPCs with PMPs treatment induced a decrease of all investigated platelet molecules, however not comparable with that recorded when EPC individual treatment was applied.

Conclusion

EPCs have the ability to reduce platelet activation and to modulate their pro-inflammatory and anti-thrombogenic properties in hypertension associated with hypercholesterolemia. Although, PMPs have several beneficial effects in combination with EPCs, these did not improve the EPC effects. These findings reveal a new biological role of circulating EPCs in platelet function regulation, and may contribute to understand their cross talk, and the mechanisms of atherosclerosis.     

Monocyte Chemoattractant Protein-1 Deficiency Attenuates Oxidative Stress and Protects against Ovariectomy-Induced Chronic Inflammation in Mice

Background

Loss of ovarian function is highly associated with an elevated risk of metabolic disease. Monocyte chemoattractant protein-1 (MCP-1, C-C chemokine ligand 2) plays critical roles in the development of inflammation, but its role in ovariectomy (OVX)-induced metabolic disturbance has not been known.

Methodology and Principal Findings

We investigated the role of MCP-1 in OVX-induced metabolic perturbation using MCP-1-knockout mice. OVX increased fat mass, serum levels of MCP-1, macrophage-colony stimulating factor (M-CSF), and reactive oxygen species (ROS), whereas MCP-1 deficiency attenuated these. OVX-induced increases of visceral fat resulted in elevated levels of highly inflammatory CD11c-expressing cells as well as other immune cells in adipose tissue, whereas a lack of MCP-1 significantly reduced all of these levels. MCP-1 deficiency attenuated activation of phospholipase Cγ2, transforming oncogene from Ak strain, and extracellular signal-regulated kinase as well as generation of ROS, which is required for up-regulating CD11c expression upon M-CSF stimulation in bone marrow-derived macrophages.

Conclusions/Significance

Our data suggested that MCP-1 plays a key role in developing metabolic perturbation caused by a loss of ovarian functions through elevating CD11c expression via ROS generation.     

FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing

Background

Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo.

Methodology and Principal Findings

To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression.

Conclusions

In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.     

Effects of Mechanical Force on Cytoskeleton Structure and Calpain-Induced Apoptosis in Rat Dorsal Root Ganglion Neurons In Vitro

Background

A sudden mechanical insult to the spinal cord is usually caused by changing pressure on the surface of the spinal cord. Most of these insults are mechanical force injuries, and their mechanism of injury to the spinal cord is largely unknown.

Methods

Using a compression-driven instrument to simulate mechanical force, we applied mechanical pressure of 0.5 MPa to rat dorsal root ganglion (DRG) neurons for 10 min to investigate cytoskeletal alterations and calpain-induced apoptosis after the mechanical force injury.

Results

The results indicated that mechanical forces affect the structure of the cytoskeleton and cell viability, induce early apoptosis, and affect the cell cycle of DRG neurons. In addition, the calpain inhibitor PD150606 reduced cytoskeletal degradation and the rate of apoptosis after mechanical force injury.

Conclusion

Thus, calpain may play an important role in DRG neurons in the regulation of apoptosis and cytoskeletal alterations induced by mechanical force. Moreover, cytoskeletal alterations may be substantially involved in the mechanotransduction process in DRG neurons after mechanical injury and may be induced by activated calpain. To our knowledge, this is the first report to demonstrate a relationship between cytoskeletal degradation and apoptosis in DRG neurons.     

MCP/CCR2 Signaling Is Essential for Recruitment of Mesenchymal Progenitor Cells during the Early Phase of Fracture Healing

Objective

The purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models.

Methods

The expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1^−/− and C-C chemokine receptor 2 (CCR2)^−/− mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1^−/− mice and were evaluated by histological staining and micro-CT.

Results

MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1^−/− and CCR2^−/− mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1^−/− mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1^−/− mouse. Conversely, transplantation of MCP-1^−/− mouse-derived grafts into host WT mice markedly decreased new bone formation.

Conclusions

MCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.     

Urinary Concentration of Monocyte Chemoattractant Protein-1 in Idiopathic Glomerulonephritis: A Long-Term Follow-Up Study

Background

Monocyte chemoattractant protein-1 (MCP-1), which is up regulated in kidney diseases, is considered a marker of kidney inflammation. We examined the value of urine MCP-1 in predicting the outcome in idiopathic glomerulonephritis.

Methods

Between 1993 and 2004, 165 patients (68 females) diagnosed with idiopathic proteinuric glomerulopathy and with serum creatinine <150 µmol/L at diagnosis were selected for the study. Urine concentrations of MCP-1 were analyzed by ELISA in early morning spot urine samples collected on the day of the diagnostic kidney biopsy. The patients were followed until 2009. The progression rate to end-stage kidney disease was calculated using Kaplan–Meier survival analysis. End-stage kidney disease (ESKD) was defined as the start of kidney replacement therapy during the study follow-up time.

Results

Patients with proliferative glomerulonephritis had significantly higher urinary MCP-1 excretion levels than those with non-proliferative glomerulonephritis (p<0.001). The percentage of patients whose kidney function deteriorated significantly was 39.0% in the high MCP-1 excretion group and 29.9% in the low MCP-1 excretion group. However, after adjustment for confounding variables such as glomerular filtration rate (GFR) and proteinuria, there was no significant association between urine MCP-1 concentration and progression to ESKD, (HR = 1.75, 95% CI = 0.64–4.75, p = 0.27).

Conclusion

Our findings indicate that progression to end-stage kidney disease in patients with idiopathic glomerulopathies is not associated with urine MCP-1 concentrations at the time of diagnosis.     

A Pattern of Early Radiation-Induced Inflammatory Cytokine Expression Is Associated with Lung Toxicity in Patients with Non-Small Cell Lung Cancer

Purpose

Lung inflammation leading to pulmonary toxicity after radiotherapy (RT) can occur in patients with non-small cell lung cancer (NSCLC). We investigated the kinetics of RT induced plasma inflammatory cytokines in these patients in order to identify clinical predictors of toxicity.

Experimental Design

In 12 NSCLC patients, RT to 60 Gy (30 fractions over 6 weeks) was delivered; 6 received concurrent chemoradiation (chemoRT) and 6 received RT alone. Blood samples were taken before therapy, at 1 and 24 hours after delivery of the 1^st fraction, 4 weeks into RT, and 12 weeks after completion of treatment, for analysis of a panel of 22 plasma cytokines. The severity of respiratory toxicities were recorded using common terminology criteria for adverse events (CTCAE) v4.0.

Results

Twelve cytokines were detected in response to RT, of which ten demonstrated significant temporal changes in plasma concentration. For Eotaxin, IL-33, IL-6, MDC, MIP-1α and VEGF, plasma concentrations were dependent upon treatment group (chemoRT vs RT alone, all p-values <0.05), whilst concentrations of MCP-1, IP-10, MCP-3, MIP-1β, TIMP-1 and TNF-α were not. Mean lung radiation dose correlated with a reduction at 1 hour in plasma levels of IP-10 (r² = 0.858, p<0.01), MCP-1 (r² = 0.653, p<0.01), MCP-3 (r² = 0.721, p<0.01), and IL-6 (r² = 0.531, p = 0.02). Patients who sustained pulmonary toxicity demonstrated significantly different levels of IP-10 and MCP-1 at 1 hour, and Eotaxin, IL-6 and TIMP-1 concentration at 24 hours (all p-values <0.05) when compared to patients without respiratory toxicity.

Conclusions

Inflammatory cytokines were induced in NSCLC patients during and after RT. Early changes in levels of IP-10, MCP-1, Eotaxin, IL-6 and TIMP-1 were associated with higher grade toxicity. Measurement of cytokine concentrations during RT could help predict lung toxicity and lead to new therapeutic strategies.