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Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca 2+ current density (I CaL) and steady-state inactiva-tion of I CaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMWAGEs contribute to the development of cardiovascular dysfunction in vivo, next to the wellknown low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca2+ current density (ICaL) and steady-state inactivation of ICaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in eft ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca2+ current density was significantly decreased in the HMW-AGEs group. L-type Ca2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function. Conclusion: Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome. Acknowledgements We thank Marc Jans for assisting with the EM. The authors also thank Petra Bex, Rosette Beenaerts and Kanigula Mubagwa for their skillful technical assistance. The graphical abstract was created using images from Servier Medical Art Commons Attribution 3.0 Unported License (http://smart.servier.com). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. Funding This work was supported by a Bijzonder onderzoeksfonds (BOF) grant from Hasselt University (15NI06-BOF). Statement of Ethics The animal protocol was approved by the Local Ethical Committee (Ethical Commission for Animal Experimentation, UHasselt, Diepenbeek, Belgium). All animal procedures were performed conforming to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Only trained researchers, certified with a Laboratory Animal Science course according to the Federation of European Laboratory Animal Science Associations, performed animal handling and procedures.

The apical papilla is a stem cell rich tissue located at the base of the developing dental root and is responsible for the progressive elongation and maturation of the root. The multipotent stem cells of the apical papilla (SCAP) are extensively studied in cell culture since they demonstrate a high capacity for osteogenic, adipogenic, and chondrogenic differentiation and are thus an attractive stem cell source for stem cell-based therapies. Currently, only few studies are dedicated to determining the role of the apical papilla in dental root development. In this review, we will focus on the architecture of the apical papilla and describe the specific SCAP signaling pathways involved in root maturation. Furthermore, we will explore the heterogeneity of the SCAP phenotype within the tissue and determine their micro-environmental interaction. Understanding the mechanism of postnatal dental root growth could further aid in developing novel strategies in dental root regeneration.

Growing evidence supports the role of advanced glycation end products (AGEs) in the development of diabetic vascular complications and cardiovascular diseases (CVDs). We have shown that high-molecular-weight AGEs (HMW-AGEs), present in our Western diet, impair cardiac function. Whether HMW-AGEs affect vascular function remains unknown. In this study, we aimed to investigate the impact of chronic HMW-AGEs exposure on vascular function and structure. Adult male Sprague Dawley rats were daily injected with HMW-AGEs or control solution for 6 weeks. HMW-AGEs animals showed intracardiac pressure overload, characterized by increased systolic and mean pressures. The contraction response to PE was increased in aortic rings from the HMW-AGEs group. Relaxation in response to ACh, but not SNP, was impaired by HMW-AGEs. This was associated with reduced plasma cyclic GMP levels. SOD restored ACh-induced relaxation of HMW-AGEs animals to control levels, accompanied by a reduced half-maximal effective dose (EC50). Finally, collagen deposition and intima-media thickness of the aortic vessel wall were increased with HMW-AGEs. Our data demonstrate that chronic HMW-AGEs exposure causes adverse vascular remodelling. This is characterised by disturbed vasomotor function due to increased oxidative stress and structural changes in the aorta, suggesting an important contribution of HMW-AGEs in the development of CVDs.

Background and Purpose Up to 50-60% of all cancer patients receive radiotherapy as part of their treatment strategy. However, the mechanisms accounting for increased vascular risks after irradiation are not completely understood. Mitochondrial dysfunction has been identified as a potential cause of radiation-induced atherosclerosis. Materials and Methods Assays for apoptosis, cellular metabolism, mitochondrial DNA content, functionality and morphology were used to compare the response of endothelial cells to a single 2 Gy dose of X-rays under basal conditions or after pharmacological treatments that either reduced (EtBr) or increased (rosiglitazone) mitochondrial content. Results Exposure to ionizing radiation caused a persistent reduction in mitochondrial content of endothelial cells. Pharmacological reduction of mitochondrial DNA content rendered endothelial cells more vulnerable to radiation-induced apoptosis, whereas rosiglitazone treatment increased oxidative metabolism and redox state and decreased the levels of apoptosis after irradiation. Conclusion Pre-existing mitochondrial damage sensitizes endothelial cells to ionizing radiation-induced mitochondrial dysfunction. Rosiglitazone protects endothelial cells from the detrimental effects of radiation exposure on mitochondrial metabolism and oxidative stress. Thus, our findings indicate that rosiglitazone may have potential value as prophylactic for radiation-induced atherosclerosis. This work was funded by EU FP7 DoReMi network of excellence (grant #249689), EU FP7 project ProCardio (grant #295823),the Belgian Federal Agency for Nuclear Control FANC-AFCN (grant #CO-90-13-3289-00) and the Belgian Fonds National de la Recherche Scientifique (F.R.S.-FNRS). BB, NB, and EC are supported by a doctoral SCK CEN grant. PS is a F.R.S.-FNRS Senior Research Associate. Aerts, A (reprint author), Belgian Nucl Res Ctr SCK CEN, Inst Environm Hlth & Safety, Radiobiol Unit, Mol, Belgium. an.aerts@sckcen.be

Fibroblast activation protein-alpha (FAP alpha) is a membrane protein with dipeptidyl-peptidase and type I collagenase activity and is expressed during fetal growth. At the age of adolescence, FAP alpha expression is greatly reduced, only emerging in pathologies associated with extracellular matrix remodeling. We determined whether FAP alpha is expressed in human dental tissue involved in root maturation i.e., dental follicle and apical papilla and in dental pulp tissue. The dental follicle revealed a high concentration of FAP alpha and vimentin-positive cells within the stromal tissue. A similar observation was made in cell culture and FACS analysis confirmed these as dental follicle stem cells. Within the remnants of the Hertwigs' epithelial root sheath, we observed FAP alpha staining in the E-cadherin positive and vimentin-negative epithelial islands. FAP alpha- and vimentin-positive cells were encountered at the periphery of the islands suggesting an epithelial mesenchymal transition process. Analysis of the apical papilla revealed two novel histological regions; the periphery with dense and parallel aligned collagen type I defined as cortex fibrosa and the inner stromal tissue composed of less compacted collagen defined as medulla. FAP alpha expression was highly present within the medulla suggesting a role in extracellular matrix remodeling. Dental pulp tissue uncovered a heterogeneous FAP alpha staining but strong staining was noted within odontoblasts.In vitrostudies confirmed the presence of FAP alpha expression in stem cells of the apical papilla and dental pulp. This study identified the expression of FAP alpha expression in dental stem cells which could open new perspectives in understanding dental root maturation and odontoblast function. FUNDING TV (GOC1916N), PH (12D8516N), PG (12U7718N), and JR (G089213N) were supported by the Research FoundationFlanders (FWO-Vlaanderen). ACKNOWLEDGMENTS The authors thank Marc Jans for processing and sectioning of araldite embedded samples, Jeanine Santermans for embedding and sectioning of paraffin sections, and Ana Amaya Garrido for her valuable contribution to the experiments. Driesen, RB (corresponding author), Hasselt Univ, Fac Med, Diepenbeek, Belgium ; Hasselt Univ, Biomed Res Inst, Lab Morphol, Diepenbeek, Belgium. ronald.driesen@uhasselt.be

Background Mechanical right ventricular (RV) support in pulmonary arterial hypertension patients has been feared to cause pulmonary hemorrhage and to be detrimental for the after-load–sensitive RV. Continuous low-flow pumps offer promise but remain insufficiently tested. Methods The pulmonary artery was banded in 20 sheep in this study. Eight weeks later, a Synergy micro-pump (HeartWare International, Framingham MA) was inserted in 10 animals, driving blood from the right atrium to the pulmonary artery. After magnetic resonance imaging, hemodynamics and RV pressure–volume loop data were recorded. Eight weeks later, RV function was assessed in the same way, followed by histologic analysis of the ventricular tissue. Results During the 8 weeks of support, RV volumes and central venous pressure decreased significantly, whereas RV contractility increased. Pulmonary artery pressure increased modestly, particularly its diastolic component. RV contribution to total right-sided cardiac output increased from 12 ± 12% to 41 ± 9% ( p ). After pump inactivation, and compared with 8 weeks earlier, RV volumes had significantly decreased, tricuspid valve regurgitation had almost disappeared, and RV contractility had significantly increased, resulting in significantly increased RV forward power (0.25 ± 0.05 vs 0.16 ± 0.06 W, p = 0.014). Fulton index and RV myocyte size were significantly smaller, and without changes in fibrosis, when compared with controls. Conclusions Prolonged continuous low-flow RV mechanical support significantly unloads the chronic pressure–overloaded RV and improves cardiac output. After 8 weeks, RV hemodynamic recovery and reverse remodeling begin to occur, without increased fibrosis.

Dental pulp is a highly vascularized and innervated tissue containing a heterogeneous stem cell population with multilineage differentiation potential. Current endodontic treatments focus on the preservation of the pulp tissue and the regeneration of dental pulp after pathological insults. Human dental pulp stem cells (hDPSCs) are currently investigated as stem cell-based therapy for pulp regeneration and for peripheral nerve injury in which neurons and Schwann cells display limited regenerative capacity. We have developed a neuronal differentiation protocol for hDPSCs that requires neurosphere formation before neuronal maturation. Moreover, Schwann cell differentiation of hDPSCs in our group revealed that differentiated hDPSCs have acquired the ability to myelinate and guide neurites from dorsal root ganglia. Besides their dynamic differentiation capacity, hDPSCs were shown to exert a paracrine effect on neural and endothelial cells. Analysis of hDPSC conditioned medium revealed the secretion of a broad spectrum of growth factors including brain-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, and glial-derived neurotrophic factor. Application of the conditioned medium to endothelial cells promoted cell migration and tubulogenesis, indicating a paracrine proangiogenic effect. This hypothesis was enforced by the enhanced formation of blood vessels in the chorioallantoic membrane assay in the presence of hDPSCs. In addition, transplantation of 3-dimensional-printed hydroxyapatite scaffolds containing peptide hydrogels and hDPSCs into immunocompromised mice revealed blood vessel ingrowth, pulplike tissue formation, and osteodentin deposition suggesting osteogenic/odontogenic differentiation of hDPSCs. Future studies in our research group will focus on the pulp regeneration capacity of hDPSCs and the role of fibroblasts within the pulp extracellular matrix.

BACKGROUND: Interstitial fibrosis is an important component of diastolic, and systolic, dysfunction in heart failure (HF) and depends on activation and differentiation of fibroblasts into myofibroblasts (MyoFb). Recent clinical evidence suggests that in late-stage HF, fibrosis is not reversible. OBJECTIVES: The study aims to examine the degree of differentiation of cardiac MyoFb in end-stage HF and the potential for their phenotypic reversibility. METHODS: Fibroblasts were isolated from the left ventricle of the explanted hearts of transplant recipients (ischemic and dilated cardiomyopathy), and from nonused donor hearts. Fibroblasts were maintained in culture without passaging for 4 or 8 days (treatment studies). Phenotyping included functional testing, immunostaining, and expression studies for markers of differentiation. These data were complemented with immunohistology and expression studies in tissue samples. RESULTS: Interstitial fibrosis with cross-linked collagen is prominent in HF hearts, with presence of activated MyoFbs. Tissue levels of transforming growth factor (TGF)-β1, lysyl oxidase, periostin, and osteopontin are elevated. Fibroblastic cells isolated from HF hearts are predominantly MyoFb, proliferative or nonproliferative, with mature α-smooth muscle actin stress fibers. HF MyoFb express high levels of profibrotic cytokines and the TGF-β1 pathway is activated. Inhibition of TGF-β1 receptor kinase in HF MyoFb promotes dedifferentiation of MyoFb with loss of α-smooth muscle actin and depolymerization of stress fibers, and reduces the expression of profibrotic genes and cytokines levels to non-HF levels. CONCLUSION: MyoFb in end-stage HF have a variable degree of differentiation and retain the capacity to return to a less activated state, validating the potential for developing antifibrotic therapy targeting MyoFb. ispartof: JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY vol:73 issue:18 pages:2267-2282 ispartof: location:United States status: published

After myocardial infarction, resident fibroblasts (Fb) differentiate towards myofibroblasts (MyoFb), generating the scar tissue and the interstitial fibrosis seen in the adjacent myocardium. Fb and MyoFb have the potential to interact with cardiac myocytes (CMs) but insight into the phenotype-specific role and mode of interaction is still incomplete. Our objectives are to further define the modulation of CMs by MyoFbs compared to Fbs, as well as the role of direct contact through gap junctions vs. soluble mediators, using Fbs and CMs from pig left ventricle. Fbs were treated to maintain an undifferentiated state (SD-208) or to attain full differentiation to MyoFb (TGF-β1). Fbs and MyoFbs were co-cultured with CMs, with the possibility of direct contact or separated by a Thincert membrane. Only in direct co-culture, both Fbs and MyoFbs were able to decrease CM viability after 2 days. Only MyoFbs induced significant distal spreading of CMs in both direct and indirect co-culture. MyoFbs, but not Fbs, readily made connections with CMs in direct co-culture and connexin 43 expression in MyoFb was higher than in Fb. When coupled to CMs, MyoFbs reduced the CM action potential duration and hyperpolarized the CM resting membrane potential. Uncoupling reversed these effects. In conclusion, MyoFbs, but not Fbs, alter the CM structural phenotype. MyoFbs, but not Fbs, are likely to electrically connect to CMs and thereby modulate the CM membrane potential. These data provide further support for an active role of MyoFbs in the arrhythmogenic substrate after cardiac remodelling. ispartof: SCIENTIFIC REPORTS vol:9 issue:1 ispartof: location:England status: published

Introduction Compelling evidence pinpoints that pulp tissue engineering after the transplantation of stem cells is possible. Although intriguing, severe problems regarding clinical feasibility remain. Cell homing has been proposed as a viable alternative in which dentin-derived growth factors in a conducive scaffold may attract resident cells to form pulplike tissue. In this study, an ectopic animal model for in situ dental pulp tissue engineering was developed to evaluate whether pulplike tissue formation in empty root canals after the attraction of stem cells was possible and whether this could be enhanced by dentin-derived growth factors. Methods Three types of fibrin (custom-made fibrin, fibrin sealant, and plasma rich in growth factors [PRGF]) as well as a self-assembling peptide were evaluated in vivo in a modified tooth root model using human teeth. Root canal dentin was conditioned with EDTA, tooth roots were filled with growth factor–laden scaffolds, and dental pulp stem cells in collagen were placed at the root tip. Constructs were implanted into immunocompromised mice for 4 weeks and subsequently analyzed histologically. Differential interference contrast and second harmonic generation imaging were performed for selected sections. Results For custom-made fibrin and fibrin sealant with dentin matrix proteins, migration into the roots and the formation of a pulplike tissue were observed, whereas the peptide-based scaffold appeared less suitable. PRGF supported tissue formation regardless of the addition of dentin matrix proteins. In the test groups with dentin matrix proteins and EDTA conditioning, differentiated odontoblastlike cells extended cellular processes into the dentinal tubules, which coincided with the deposition of the newly formed collagenous dentin matrix. Conclusions This new cell homing model provides evidence that fibrin derivatives make applicable scaffolds and that dentin-derived proteins induce chemotaxis and pulplike tissue formation.

Advanced glycation end products (AGEs) play a key role in the progression of heart failure. Whether treatments limiting AGEs formation would prevent adverse left ventricular remodeling after myocardial infarction (MI) remain unknown. We investigated whether pyridoxamine (PM) could limit adverse cardiac outcome in MI. Rats were divided into MI, MI + PM and Sham. Echocardiography and hemodynamic parameters were used to assess cardiac function 8 weeks post-surgery. Total interstitial collagen, collagen I and collagen III were quantified using Sirius Red and polarized light microscopy. PM improved survival following LAD occlusion. Pre-treatment with PM significantly decreased the plasma AGEs levels. MI rats treated with PM displayed reduced left ventricular end-diastolic pressure and tau compared to untreated MI rats. Deformation parameters were also improved with PM. The preserved diastolic function was related to the reduced collagen content, in particular in the highly cross-linked collagen type I, mainly in the peri-infarct region, although not via TGF-beta 1 pathway. Our data indicate that PM treatment prevents the increase in AGEs levels and reduces collagen levels in a rat model of MI, resulting in an improved cardiac phenotype. As such, therapies targeting formation of AGEs might be beneficial in the prevention and/or treatment of maladaptive remodeling following MI. Belgian Science Policy (IAP VII network) [P7/10]; UHasselt BOF-NI [15NI06-BOF]

Differentiation of foetal cardiomyocytes is accompanied by sequential actin isoform expression, i.e. down-regulation of the 'embryonic' alpha smooth muscle actin, followed by an up-regulation of alpha skeletal actin (alphaSKA) and a final predominant expression of alpha cardiac actin (alphaCA). Our objective was to detect whether re-expression of alphaSKA occurred during cardiomyocyte dedifferentiation, a phenomenon that has been observed in different pathologies characterized by myocardial dysfunction. Immunohistochemistry of alphaCA, alphaSKA and cardiotin was performed on left ventricle biopsies from human patients after coronary bypass surgery. Furthermore, actin isoform expression was investigated in left ventricle samples of rabbit hearts suffering from pressure- and volume-overload and in adult rabbit ventricular cardiomyocytes during dedifferentiation in vitro. Atrial goat samples up to 16 weeks of sustained atrial fibrillation (AF) were studied ultrastructurally and were immunostained for alphaCA and alphaSKA. Up-regulation of alphaSKA was observed in human ventricular cardiomyocytes showing down-regulation of alphaCA and cardiotin. A patchy re-expression pattern of alphaSKA was observed in rabbit left ventricular tissue subjected to pressure- and volume-overload. Dedifferentiating cardiomyocytes in vitro revealed a degradation of the contractile apparatus and local re-expression of alphaSKA. Comparable alphaSKA staining patterns were found in several areas of atrial goat tissue during 16 weeks of AF together with a progressive glycogen accumulation at the same time intervals. The expression of alphaSKA in adult dedifferentiating cardiomyocytes, in combination with PAS-positive glycogen and decreased cardiotin expression, offers an additional tool in the evaluation of myocardial dysfunction and indicates major changes in the contractile properties of these cells.

Scarring and remodeling of the left ventricle (LV) after myocardial infarction (MI) results in ischemic cardiomyopathy with reduced contractile function. Regional differences related to persisting ischemia may exist. We investigated the hypothesis that mitochondrial function and structure is altered in the myocardium adjacent to MI with reduced perfusion (MIadjacent) and less so in the remote, nonischemic myocardium (MIremote). We used a pig model of chronic coronary stenosis and MI ( n = 13). Functional and perfusion MR imaging 6 wk after intervention showed reduced ejection fraction and increased global wall stress compared with sham-operated animals (Sham; n = 14). Regional strain in MIadjacent was reduced with reduced contractile reserve; in MIremote strain was also reduced but responsive to dobutamine and perfusion was normal compared with Sham. Capillary density was unchanged. Cardiac myocytes isolated from both regions had reduced basal and maximal oxygen consumption rate, as well as through complex I and II, but complex IV activity was unchanged. Reduced respiration was not associated with detectable reduction of mitochondrial density. There was no significant change in AMPK or glucose transporter expression levels, but glycogen content was significantly increased in both MIadjacent and MIremote. Glycogen accumulation was predominantly perinuclear; mitochondria in this area were smaller but only in MIadjacent where also subsarcolemmal mitochondria were smaller. In conclusion, after MI reduction of mitochondrial respiration and glycogen accumulation occur in all LV regions suggesting that reduced perfusion does not lead to additional specific changes and that increased hemodynamic load is the major driver for changes in mitochondrial function.

Background Cardiotin expression is observed in adult cardiac tissue. In the present study, we provide evidence for the specific localization of cardiotin in cardiac mitochondria and for its down-regulation during adaptive remodeling (dedifferentiation) of cardiomyocytes. Methods Immunocytochemistry was used to study cardiotin localization in adult rabbit papillary muscle, in late-stage embryonic rabbit left ventricular tissue, and in left ventricle samples of rabbits suffering from pressure and volume overload. Western blot analysis of cardiotin was performed in purified pig heart mitochondrial fractions. Cardiotin expression was monitored in vitro in isolated adult rat and rabbit left ventricular cardiomyocytes. Results Western blot analysis revealed the presence of cardiotin in the mitochondrial fractions of pig heart. Immunoelectron microscopy confirmed the presence of cardiotin in cardiac mitochondria of normal adult rabbits both in vivo and in vitro. Quantification of the localization of immunogold particles suggests an association of cardiotin with the mitochondrial inner membrane. Cardiotin expression is initiated in late-stage embryonic rabbit heart, whereas in adult ventricular tissue cardiotin clearly stained longitudinal arrays of mitochondria. Pressure- and volume-overloaded myocardium showed a reduction in cardiotin expression in dispersed local myocardial areas. Cell cultures of adult cardiomyocytes showed a gradual loss in cardiotin expression in parallel with a sarcomeric remodeling. Conclusions Our results demonstrate the specific localization of cardiotin in adult cardiomyocyte mitochondria and propose its use as an early marker for cardiomyocyte adaptive remodeling and dedifferentiation.