Biomarkers of inflammation and innate immunity in atrophic nonunion fracture.

BACKGROUND: Nonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing. METHODS: Thirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF-MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann-Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC-MS/MS, nephelometry, western blotting or ELISA assays. RESULTS: From the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF-MS using CM10 and IMAC-Cu(2+) ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin ß subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls. CONCLUSION: Two proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.

Decreased pool of mesenchymal stem cells is associated with altered chemokines serum levels in atrophic nonunion fractures.

Nonunion fractures can cause severe dysfunction and are often difficult to treat mainly due to a poor understanding of their physiopathology. Although many aspects of impaired fracture healing have been extensively studied, little is known about the cellular and molecular mechanisms leading to atrophic nonunion. Therefore, the aim of the present study was to assess the pools and biological functions of bone marrow-derived mesenchymal stem cells (hMSCs) and circulating endothelial progenitor cells (EPCs) in atrophic nonunion patients compared to healthy subjects, and the systemic levels of growth factors involved in the recruitment, proliferation and differentiation of these cells. In nonunions, the pool of hMSCs was decreased and their proliferation delayed. However, once committed, hMSCs from nonunions were able to proliferate, differentiate into osteoblastic cells and mineralize in vitro as efficiently as hMSCs from healthy subjects. In parallel, we found altered serum levels of chemokines and growth factors involved in the chemotaxis and proliferation of hMSCs such as leptin, interleukin-6 (IL-6) and its soluble receptor, platelet-derived growth factor-BB (PDGF-BB), stem cell factor (SCF) and insulin-like growth factor-1 (IGF-1). Moreover, we showed that the number of EPCs and their regulating growth factors were not affected in nonunion patients. If nonunion is generally attributed to a vascular defect, our results also support a role for a systemic mesenchymal and osteogenic cell pool defect that might be related to alterations in systemic levels of factors implicated in their chemotaxis and proliferation.

Dipeptidyl peptidase IV inhibition improves cardiorenal function in overpacing-induced heart failure.

AIMS: Recent studies indicate that brain natriuretic peptide (BNP(1-32)) may be truncated into BNP(3-32) by dipeptidyl peptidase IV (DPP4) and that BNP(3-32) has reduced biological activities compared with BNP(1-32). We investigated if DPP4 contributes to the cardiorenal alterations and to the attenuated response to BNP seen in heart failure. METHODS AND RESULTS: Haemodynamic and renal assessment was performed in 12 pigs at baseline, 4 weeks after pacing-induced heart failure, and during BNP infusion. They were randomized to either placebo or treatment with a DPP4 inhibitor, sitagliptin. After 4 weeks of pacing, heart rate was reduced compared with baseline in the sitagliptin group (60 ± 2 vs. 95 ± 16 b.p.m., P < 0.01), and an increase in stroke volume was observed in the sitagliptin group compared with placebo (+24 ± 6% vs. -17 ± 7%, P < 0.01). Glomerular filtration rate declined at week 4 compared with baseline in the placebo group (1.3 ± 0.4 vs. 2.3 ± 0.3 mL/kg/min, P < 0.01) but remained preserved in the sitagliptin group [1.8 ± 0.2 vs. 2.0 ± 0.3 mL/kg/min, P = NS (non-significant)]. In the sitagliptin group, BNP infusion improved end-systolic elastance (68 ± 5 vs. 31 ± 4 mmHg/kg/mL, P < 0.05), ventricular-arterial coupling, and mechanical efficiency. Compared with controls (n = 6), myocardial gene expression of BNP, interleukin-6, Na(+)-Ca(2+) exchanger, and calmodulin was up-regulated in the placebo group, but not in the sitagliptin group. CONCLUSION: In pacing-induced heart failure, DPP4 inhibition preserves the glomerular filtration rate, modulates stroke volume and heart rate, and potentiates the positive inotropic effect of exogenous BNP at no energy expense.

Evaluation of the influence of age on pulmonary arterial pressure by use of right ventricular catheterization, pulsed-wave Doppler echocardiography, and pulsed-wave tissue Doppler imaging in healthy Beagles.

OBJECTIVE: To assess the influence of age on pulmonary hemodynamics and hemorheological properties in healthy dogs. ANIMALS: 14 healthy Beagles. PROCEDURES: Dogs were placed in 2 age groups as follows: young dogs (or= 8 years old; 6). Hematologic characteristics, plasma total protein and fibrinogen concentrations, and blood viscosity were measured. Systolic time intervals of pulmonary blood flow were recorded by pulsed-wave Doppler echocardiography. Early (E') and late (A') diastolic myocardial velocities, isovolumic contraction velocity, and systolic myocardial velocity of the free tricuspid annulus were recorded by pulsed-wave tissue Doppler imaging (TDI). Dogs were anesthetized and pulmonary arterial pressures (PAP) and cardiac output were recorded with a pediatric thermodilution Swan-Ganz catheter. RESULTS: Hemorheological measurements were not different between the 2 groups. Systolic, mean, and diastolic PAP were higher in old dogs, compared with values in young dogs; this difference was attributed to a high pulmonary vascular resistance and low arterial compliance in old dogs. Systolic time intervals of pulmonary blood flow stayed unchanged. The A' wave of the free tricuspid annulus was increased in old, compared with that young dogs, and the E':A' ratio was decreased. Pulmonary vascular resistance was inversely correlated with compliance. CONCLUSIONS AND CLINICAL RELEVANCE: The age of dogs should be taken into account when interpreting pulmonary hemodynamic results and TDI variables of right ventricular diastolic function.

Ventricular-arterial uncoupling in heart failure with preserved ejection fraction after myocardial infarction in dogs - invasive versus echocardiographic evaluation.

BACKGROUND: Heart failure with preserved left ventricular ejection fraction and abnormal diastolic function is commonly observed after recovery from an acute myocardial infarction. The aim of this study was to investigate the physiopathology of heart failure with preserved ejection fraction in a model of healed myocardial infarction in dogs. METHODS: Echocardiography, levels of neurohormones and conductance catheter measurements of left ventricular pressure-volume relationships were obtained in 17 beagle dogs 2 months after a coronary artery ligation, and in 6 controls. RESULTS: Healed myocardial infarction was associated with preserved echocardiographic left ventricular ejection fraction (0.57 +/- 0.01, mean +/- SEM) and altered Doppler mitral indices of diastolic function. NT-proBNP was increased, aldosterone was decreased, and norepinephrine was unchanged. Invasive measurements showed a markedly decreased end-systolic elastance (2.1 +/- 0.2 vs 6.1 +/- 0.8, mmHg/ml, p < 0.001) and end-systolic elastance to effective arterial elastance ratio (0.6 +/- 0.1 vs 1.4 +/- 0.2, p < 0.001), with altered active relaxation (dP/dtmin -1992 +/- 71 vs -2821 +/- 305, mmHg/s, p < 0.01) but preserved left ventricular capacitance (70 +/- 6 vs 61 +/- 3, ml at 20 mmHg, p = NS) and stiffness constant. Among echocardiographic variables, the wall motion score index was the most reliable indicator of cardiac contractility while E', E/A and E'/A' were correlated to dP/dtmin. CONCLUSIONS: In the canine model of healed myocardial infarction induced by coronary ligation, heart failure is essentially characterized by an altered contractility with left ventricular-arterial uncoupling despite vascular compensation rather than by abnormal diastolic function.

Cardiac insulin-like growth factor-1 and cyclins gene expression in canine models of ischemic or overpacing cardiomyopathy.

BACKGROUND: Insulin-like growth factor-1 (IGF-1), transforming growth factor beta (TGFbeta) and cyclins are thought to play a role in myocardial hypertrophic response to insults. We investigated these signaling pathways in canine models of ischemic or overpacing-induced cardiomyopathy. METHODS: Echocardiographic recordings and myocardial sampling for measurements of gene expressions of IGF-1, its receptor (IGF-1R), TGFbeta and of cyclins A, B, D1, D2, D3 and E, were obtained in 8 dogs with a healed myocardial infarction, 8 dogs after 7 weeks of overpacing and in 7 healthy control dogs. RESULTS: Ischemic cardiomyopathy was characterized by moderate left ventricular systolic dysfunction and eccentric hypertrophy, with increased expressions of IGF-1, IGF-1R and cyclins B, D1, D3 and E. Tachycardiomyopathy was characterized by severe left ventricular systolic dysfunction and dilation with no identifiable hypertrophic response. In the latter model, only IGF-1 was overexpressed while IGF-1R, cyclins B, D1, D3 and E stayed unchanged as compared to controls. The expressions of TGFbeta, cyclins A and D2 were comparable in the 3 groups. The expression of IGF-1R was correlated with the thickness of the interventricular septum, in systole and diastole, and to cyclins B, D1, D3 and E expression. CONCLUSION: These results agree with the notion that IGF-1/IGF-1R and cyclins are involved in the hypertrophic response observed in cardiomyopathies.

Cell therapy with autologous bone marrow mononuclear stem cells is associated with superior cardiac recovery compared with use of nonmodified mesenchymal stem cells in a canine model of chronic myocardial infarction.

OBJECTIVE: Stem cell therapy can facilitate cardiac repair in infarcted myocardium, but the optimal cell type remains uncertain. We conducted a randomized, blind, and placebo-controlled comparison of autologous bone marrow mononuclear cell and mesenchymal stem cell therapy in a large-animal model of chronic myocardial infarction. METHODS: Eleven weeks after coronary ligation, 24 dogs received intramyocardial injections of mononuclear cells (227.106 +/- 32.106 cells), mesenchymal stem cells (232.106 +/- 40.106 cells), or placebo (n = 8 per group). Cardiac performance and remodeling were assessed up to 16 weeks' follow-up. RESULTS: At echocardiographic analysis, the wall motion score index showed a sustained improvement after mononuclear cell transfer (from 1.8 +/- 0.1 to 1.5 +/- 0.07) and a moderate late improvement after mesenchymal stem cell transfer (from 1.9 +/- 0.08 to 1.7 +/- 0.1). After mononuclear cell transfer, end-systolic elastance increased (from 2.23 +/- 0.25 to 4.42 +/- 0.55 mm Hg/mL), infarct size decreased (from 13% +/- 0.67% to 10% +/- 1.17%), N-terminal B-type natriuretic propeptide level decreased (from 608 +/- 146 to 353 +/- 118 pmol/L), and relative wall area and arterial density increased. Vascular endothelial growth factor receptor 2 expression was upregulated in the border zone. No change in cardiac contractility or histologic parameters was noted in the mesenchymal stem cell group. CONCLUSION: In a canine model of chronic myocardial infarction, bone marrow mononuclear cell transfer is superior to mesenchymal stem cell transfer in improvement of cardiac contractility and regional systolic function and reduction in infarct size and plasma N-terminal B-type natriuretic propeptide level. Functional improvement is associated with a favorable angiogenic environment and neovascularization.

Ventricular ErbB2/ErbB4 activation and downstream signaling in pacing-induced heart failure.

The neuregulin-1 (NRG-1)/ErbB system has emerged as a cardioprotective system that becomes activated during myocardial stress, most convincingly shown in response to cardiotoxic chemotherapy. Direct evidence of increased ventricular ErbB receptor activity in heart failure unrelated to cardiotoxic drugs is, however, limited. We investigated changes in NRG-1 expression, ErbB receptor phosphorylation and downstream activation of intracellular ErbB targets during rapid pacing and progressive ventricular dysfunction in the dog. Heart failure was induced in dogs by 7 weeks of rapid pacing. Ventricular function was assessed by echocardiography. Messenger RNA expression was investigated in ventricular biopsies using quantitative PCR. Activation of NRG-1/ErbB signaling and of downstream targets was investigated using immunoprecipitation and/or Western blotting. Over the course of 7 weeks of pacing and ventricular dilatation, ventricular levels of NRG-1, but not of other ErbB4 ligands, and of ADAM19, a protease promoting NRG-1 release, progressively increased. In parallel, levels of activated ErbB2 and ErbB4, phosphorylated at tyrosine residues 877/1248 and 1284 respectively, became progressively higher. Similarly, levels of total and phosphorylated PI-3 kinase increased. Surprisingly, however, and in contrast with activation of downstream targets of ErbB receptors in normal hearts, Akt and ERK1/2, remained inactivated. This study shows that ventricular ErbB2 and ErbB4 receptors become activated during the development of pacing-induced heart failure, but that downstream signaling is, at least partly, abrogated. Abrogation of cardioprotective signaling after ErbB activation is an unanticipated phenomenon in the progression of heart failure with possibly major pathophysiological significance. The underlying mechanisms should be further elucidated.

Activin-A, transforming growth factor-beta, and myostatin signaling pathway in experimental dilated cardiomyopathy.

BACKGROUND: The pathogenic mechanisms of dilated cardiomyopathy are still uncertain. A number of cytokines and growth factors participate in the remodeling process of the disease. METHODS: We investigated the cardiac myostatin, transforming growth factor (TGF)beta, and activin-A/Smad growth inhibitory signaling pathway in experimental dilated cardiomyopathy. Transvenous endomyocardial biopsies of the interventricular septum were taken weekly in 15 beagle dogs during the development of heart failure (HF) induced by rapid pacing over a period of 7 weeks. Genes involved in the myostatin-TGFbeta-activin-A/Smad signaling pathway and the cardiac hypertrophic process were quantified by real-time quantitative polymerase chain reaction. Left ventricular volume, function, and mass were evaluated by echocardiography. RESULTS: Overpacing was associated with increased left ventricular volumes and decreased ejection fraction, whereas the left ventricular mass remained unchanged. TGFbeta was increased in moderate HF. Activin-A mRNA expression was 4-fold higher in overt congestive HF than at baseline. A 2-fold decrease of activin type II receptors and activin receptor interacting protein 2 gene expressions were observed, as well as a transient decrease of follistatin. Activin type I receptors, activin receptor interacting protein 1, follistatin-related gene, and myostatin remained unchanged. The inhibitory Smad 7, a negative feedback loop regulator of the Smad pathway, was overexpressed in severe HF. Gene expression of the cyclin-dependent kinase inhibitor p21, a direct target gene of the Smad pathway, was 8-fold up-regulated in HF, whereas cyclin D1 was down-regulated. CONCLUSION: We conclude that tachycardia-induced dilated cardiomyopathy is characterized by gene overexpression of the TGFbeta-activin-A/Smad signaling pathway and their target gene p21 and by the absence of ventricular hypertrophy.

Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium.

The in vivo cardiac differentiation and functional effects of unmodified adult bone marrow mesenchymal stem cells (MSCs) after myocardial infarction (MI) is controversial. We postulated that ex vivo pretreatment of autologous MSCs using cardiomyogenic growth factors will lead to cardiomyogenic specification and will result in superior biological and functional effects on cardiac regeneration of chronically infarcted myocardium. We used a chronic dog MI model generated by ligation of the coronary artery (n = 30). Autologous dog bone marrow MSCs were isolated, culture expanded, and specified into a cardiac lineage by adding growth factors, including basic FGF, IGF-1, and bone morphogenetic protein-2. Dogs underwent cell injection >8 wk after the infarction and were randomized into two groups. Group A dogs (n = 20) received MSCs specified with growth factors (147 +/- 96 x 10(6)), and group B (n = 10) received unmodified MSCs (168 +/- 24 x 10(6)). After the growth factor treatment, MSCs stained positive for the early muscle and cardiac markers desmin, antimyocyte enhancer factor-2, and Nkx2-5. In group A dogs, prespecified MSCs colocalized with troponin I and cardiac myosin. At 12 wk, group A dogs showed a significantly larger increase in regional wall thickening of the infarcted territory (from 22 +/- 8 to 32 +/- 6% in group A; P < 0.05 vs. baseline and group B, and from 19 +/- 7 to 21 +/- 7% in group B, respectively) and a decrease in the wall motion score index (from 1.60 +/- 0.05 to 1.35 +/- 0.03 in group A; P < 0.05 vs. baseline and group B, and from 1.58 +/- 0.07 vs. 1.56 +/- 0.08 in group B, respectively). The biological ex vivo cardiomyogenic specification of adult MSCs before their transplantation is feasible and appears to improve their in vivo cardiac differentiation as well as the functional recovery in a dog model of the chronically infarcted myocardium.

Effects of ramipril on renal function during progressive overpacing-induced heart failure in dogs.

OBJECTIVE: To investigate the effects of preventive angiotensin converting enzyme inhibitor treatment with ramipril in dogs with progressively severe experimentally induced heart failure. ANIMALS: 20 dogs. PROCEDURES: Dogs were randomly allocated to receive no treatment (control) or ramipril (0.125 mg/kg, PO, daily) for 7 weeks. Physical examination, repetitive catheterization of the right side of the heart, and echocardiography were performed before the study (day 0) and weekly for 7 weeks. Renal plasma flow (RPF) as determined by para-aminohippuric acid clearance and glomerular filtration rate (GFR) as determined by creatinine and iohexol clearances were measured on day 0 and at weeks 4 and 7. RESULTS: Overpacing induced a progressive increase in right atrial pressure (RAP) and pulmonary artery pressure, occluded (PAPO), with a decrease in systemic arterial pressure. There were progressive alterations of echocardiographic indices of diastolic and systolic ventricular function. The RPF and GFR decreased before cardiac output decreased, and filtration fraction increased. The logarithm of the urinary sodium-to-potassium concentration ratio (log(10)[Na(+)/K(+)]) decreased. Significant effects of ramipril included a delay in clinical signs of heart failure, a late decrease in RAP and PAPO, and increases in the sodium excretion fraction and log(10)(Na(+)/K(+)). There was a satisfactory agreement between the creatinine and iohexol clearance measurements. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that, in this rapid-evolving, dilated cardiomyopathy, activation of the renin-angiotensin system contributes to the pathophysiology of heart failure late in the disease and essentially by an activation of renal salt and water retention.

Respiratory-related heart rate variability in progressive experimental heart failure.

Heart failure is associated with autonomic imbalance, and this can be evaluated by a spectral analysis of heart rate variability. However, the time course of low-frequency (LF) and high-frequency (HF) heart rate variability changes, and their functional correlates during progression of the disease are not exactly known. Progressive heart failure was induced in 16 beagle dogs over a 7-wk period by rapid ventricular pacing. Spectral analysis of heart rate variability and respiration, echocardiography, hemodynamic measurements, plasma atrial natriuretic factor, and norepinephrine was obtained at baseline and every week, 30 min after pacing interruption. Progressive heart failure increased heart rate (from 91 +/- 4 to 136 +/- 5 beats/min; P < 0.001) and decreased absolute and normalized (percentage of total power) HF variability from week 1 and 2, respectively (P < 0.01). Absolute LF variability did not change during the study until it disappeared in two dogs at week 7 (P < 0.05). Normalized LF variability increased in moderate heart failure (P < 0.01), leading to an increased LF-to-HF ratio (P < 0.05), but decreased in severe heart failure (P < 0.044; week 7 vs. week 5). Stepwise regression analysis revealed that among heart rate variables, absolute HF variability was closely associated with wedge pressure, right atrial and pulmonary arterial pressure, left ventricular ejection fraction and volume, ratio of maximal velocity of early (E) and atrial (A) mitral flow waves, left atrial diameter, plasma norepinephrine, and atrial natriuretic peptide (0.45 < r < 0.65, all P < 0.001). In tachycardia-induced heart failure, absolute HF heart rate variability is a more reliable indicator of cardiac dysfunction and neurohumoral activation than LF heart rate variability.

Early activation of cardiac and renal endothelin systems in experimental heart failure.

We investigated the time course of the expression of cardiac and renal endothelin systems in tachycardia-induced heart failure in dogs. Eleven beagles underwent rapid pacing at a progressively increased rate over a period of 5 wk, with a weekly clinical examination, echocardiography, measurement of circulating and urinary endothelin-1 (ET-1), and myocardial and renal tissue biopsies. Real-time quantitative PCR was used for determinations of tissue prepro-ET-1 (ppET-1), ET-1-converting enzyme (ECE-1), and ETA and ETB receptor mRNA. Cardiac and renal tissue ET-1 contents were evaluated by immunostaining and measured by radioimmunoassay at autopsy. Rapid pacing caused a progressive increase in end-systolic and end-diastolic ventricular volumes (P < 0.05) from week 2 together with a decrease in ejection fraction and in mean velocity of circumferential shortening (P < 0.05) from week 1. These changes were tightly correlated to myocardial ppET-1 and renal ETA receptor mRNA and less so to myocardial ECE-1 mRNA, and they occurred before any increase in plasma and urinary ET-1 (P < 0.05 from week 4) and clinical signs of heart failure. Renal ppET-1 did not change. Both cardiac and renal ET-1 peptide contents were increased at autopsy. We conclude that tachycardia-induced heart failure in dogs is characterized by an early activation of the cardiac and renal tissue endothelin systems, which occurs before any changes in circulating and urinary ET-1 and is closely related to altered ventricular function.