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Aims Differentiation of cardiac fibroblasts (Fbs) into myofibroblasts (MyoFbs) is responsible for connective tissue build-up in myocardial remodelling. We examined MyoFb differentiation and reversibility. Methods and results Adult rat cardiac Fbs were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different levels of Fb differentiation. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fibre formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted differentiation into α-SMA-positive MyoFb showing near the absence of proliferation, i.e. non-p-MyoFb. SD-208, a TGF-β-receptor-I (TGF-β-RI) kinase blocker, inhibited p-MyoFb differentiation as shown by stress fibre absence, low α-SMA expression, and high proliferation levels. Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and four-fold contraction. Fb produced little collagen but high levels of interleukin-10. Non-p-MyoFb had high collagen production and high monocyte chemoattractant protein-1 and tissue inhibitor of metalloproteinases-1 levels. Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation). Dedifferentiation of p-MyoFb with stress fibre de-polymerization, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress. Stress fibre de-polymerization could also be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2-day cultures in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D cultures. Conclusions Fb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile. Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Objective: Fibroblasts have been shown to couple to neonatal cardiomyocytes in heterocellular cultures through functional gap junctions. Our objective was to provide evidence for an additional type of heterocellular communication between fibroblasts and adult cardiomyocytes in vitro and in vivo. Methods: The contact areas in heterocellular co-cultures were evaluated by specific labeling and the intercellular communication was studied using preloading of fibroblasts with tracer molecules. Heterocellular fibroblast–cardiomyocyte contacts present in the in vitro setting and in the border zone of a rabbit myocardial infarction in vivo were further examined by electron microscopy. Results: Addition of fibroblasts preloaded with the fluorescent low molecular weight tracer calcein-AM to cultured myocytes indicated early dye transfer via connexin 43 functional gap junctions. At a later time-period after co-culturing, dye transfer of fibroblasts preloaded with the high molecular weight tracer dextran 10,000 suggested partial cell fusion. The membrane continuity giving rise to this partial cell fusion was confirmed by electron microscopy, clearly showing areas of intercytoplasmic contacts between fibroblasts and phenotypically adapted (dedifferentiated) cardiomyocytes. Fluorescein-labeled annexin V affinity studies revealed transient exposure of phosphatidylserine at the contact sites, suggesting that phosphatidylserine mediates the fusion process. Close contacts between cardiac fibroblasts and dedifferentiated cardiomyocytes accompanied by disruption of the basal lamina were observed in the border zone of a rabbit myocardial infarction in vivo. Conclusion: Our results suggest that the partial cell fusion-type of heterocellular communication in our co-culture model and the contacts observed in vivo may lead to new insights in cardiovascular disease. [Copyright &y& Elsevier]

Abstract: Background: Microvascular obstruction after reperfusion therapy of acute myocardial infarction is reported as an adverse promoter of left ventricular remodeling and is an important target to prevent deterioration into heart failure. In this study, we illustrate the early onset of a magnetic resonance imaged microvascular obstruction in a porcine model of acute myocardial infarction with the exact histological correlate. Methods: Occlusion of the left anterior descending coronary artery followed by 3-h reperfusion was performed in 10 pigs. Microvascular obstruction was assessed by contrast-enhanced magnetic resonance imaging (MRI). After sacrifice, serial sectioned slices of the hearts matching the MRI were stained with Triphenyl tetrazolium chloride (TTC). Biopsies were fixed, embedded in paraffin, and stained for hematoxylin–eosin. Results: Microvascular obstruction was defined with MRI as a hypoenhanced no-reflow area within the hyperenhanced infarct region. Erythrocyte plugging was consistently observed in the no-reflow area and was completely absent in the adjacent hyperenhanced infarct region. Conclusion: This model of acute ischemia–reperfusion contributes to the histological comprehension of contrast-enhanced MRI during the early stages of myocardial infarction. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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 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 left 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 < 1 × 10 –4 ). 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Within the field of tissue engineering, natural tissues are reconstructed by combining growth factors, stem cells, and different biomaterials to serve as a scaffold for novel tissue growth. As adequate vascularization and innervation are essential components for the viability of regenerated tissues, there is a high need for easily accessible stem cells that are capable of supporting these functions. Within the human tooth and its surrounding tissues, different stem cell populations can be distinguished, such as dental pulp stem cells, stem cells from human deciduous teeth, stem cells from the apical papilla, dental follicle stem cells, and periodontal ligament stem cells. Given their straightforward and relatively easy isolation from extracted third molars, dental stem cells (DSCs) have become an attractive source of mesenchymal-like stem cells. Over the past decade, there have been numerous studies supporting the angiogenic, neuroprotective, and neurotrophic effects of the DSC secretome. Together with their ability to differentiate into endothelial cells and neural cell types, this makes DSCs suitable candidates for dental tissue engineering and nerve injury repair. [ABSTRACT FROM AUTHOR]

Postconditioning and cyclosporine A prevent mitochondrial permeability transition pore opening providing cardioprotection during ischemia/reperfusion. Whether microvascular obstruction is affected by these interventions is largely unknown. Pigs subjected to coronary occlusion for 1 h followed by 3 h of reperfusion were assigned to control (n = 8), postconditioning (n = 9) or cyclosporine A intravenous infusion 10–15 min before the end of ischemia (n = 8). Postconditioning was induced by 8 cycles of repeated 30-s balloon inflation and deflation. After 3 h of reperfusion magnetic resonance imaging, triphenyltetrazolium chloride/Evans blue staining and histopathology were performed. Microvascular obstruction (MVO, percentage of gadolinium-hyperenhanced area) was measured early (3 min) and late (12 min) after contrast injection. Infarct size with double staining was smaller in cyclosporine (46.2 ± 3.1 %, P = 0.016) and postconditioning pigs (47.6 ± 3.9 %, P = 0.008) versus controls (53.8 ± 4.1 %). Late MVO was significantly reduced by cyclosporine (13.9 ± 9.6 %, P = 0.047) but not postconditioning (23.6 ± 11.7 %, P = 0.66) when compared with controls (32.0 ± 16.9 %). Myocardial blood flow in the late MVO was improved with cyclosporine versus controls (0.30 ± 0.06 vs 0.21 ± 0.03 ml/g/min, P = 0.002) and was inversely correlated with late-MVO extent (R2 = 0.93, P\0.0001). Deterioration of left ventricular ejection fraction (LVEF) between baseline and 3 h of reperfusion was smaller with cyclosporine (-7.9 ± 2.4 %, P = 0.008) but not postconditioning (-12.0 ± 5.5 %, P = 0.22) when compared with controls (-16.4 ± 5.5 %). In the three groups, infarct size (β = -0.69, P\0.001) and late MVO (β = -0.33, P = 0.02) were independent predictors of LVEF deterioration following ischemia/reperfusion (R2 = 0.73, P\0.001). Despite both cyclosporine A and postconditioning reduce infarct size, only cyclosporine A infusion had a beneficial effect on microvascular damage and was associated with better preserved LV function when compared with controls. [ABSTRACT FROM AUTHOR]

[Display omitted] • A Ti6Al4V scaffold with optimal designing parameters and mechanical properties similar to cancellous bone was 3D-printed. • To improve biocompatibility, a porous micro/nanostructured biomimetic calcium phosphate coating was applied to the scaffold. • Using the coating as a carrier, bone morphogenic protein-2 was consistently and uniformly released in a cell-mediated manner. • The bone morphogenic protein-2 integrated coating induced an even distribution of new bone formation within the scaffold. The use of Ti6Al4V in bone engineering is limited, due to the biological inertia of the surface. In this study, a porous Ti6Al4V scaffold with mechanical properties similar to cancellous bone was designed and 3D-printed. Under physiological conditions, the scaffold was immersed firstly in a 5-fold-concentrated simulated body fluid, then in a supersaturated CaP solution containing BMP-2, to form a bone-like porous micro/nano structured biomimetic coating on the surface. Scaffolds were implanted in the muscle pouches created in six beagle dogs and were retrieved four weeks later for histologic and histomorphometric analysis. Results showed that BMP-2 integrated biomimetic CaP coating induced ectopic bone formation, which was absent in other two groups. Soft tissue infiltrated the scaffold's outside 1 mm layer, while the new-formed bone was evenly distributed in the longitudinal and horizontal directions within the rest of the scaffold based on BA/TA, BIC and BA measurements. In conclusion, the BMP-2 incorporated biomimetic CaP coating creates a micro/nano surface structure on the Ti6Al4V scaffold, which helps to increase biocompatibility. The integrated BMP-2 is capable of inducing ectopic bone formation in vivo. The proposed combination may have the potential for bone reconstruction, but further studies are needed to explore its clinical applicability. [ABSTRACT FROM AUTHOR]

Fibroblast (Fb) differentiation and interstitial fibrosis contribute to cardiac remodeling and loss of function after myocardial infarction (MI). We investigated regional presence and regulation of fibrosis in a pig MI model. In vivo analysis of regional function and perfusion defined three regions: the scar, the myocardium adjacent to the scar (MIadjacent, reduced function, reduced perfusion reserve), and the remote myocardium (MIremote, minimal functional deficit, maintained perfusion). Interstitial and perivascular fibrosis, and increase of collagen type I, was only observed in the MIadjacent. Fb activated protein-alpha (FAP-α) was enriched in MIadjacent compared to MIremote. TGF-β1, which triggers Fb differentiation, was upregulated in both MIadjacent and MIremote, whereas lysyl oxidase, a regulator of collagen cross-linking, and the proteoglycans decorin and biglycan were only increased in the MIadjacent. Fb isolated and cultured for 4 days had myoFb characteristics with little difference between MIremote and MIadjacent, although RNA sequencing revealed differences in gene expression profiles. Fbs from all regions maintained proliferative capacity, and induced contraction of 3-D collagen matrices but scar myoFb was more effective. These data suggest that after MI, signaling through TGF-β1, possibly related to increased mechanical load, drives Fb activation throughout the left ventricle while regional signaling determines further maturation and extracellular matrix remodeling after MI. [ABSTRACT FROM AUTHOR]