Recombinant human bone morphogenetic protein 2 (rhBMP-2) immobilized on laser-fabricated 3D scaffolds enhance osteogenesis.

The regeneration of bone via a tissue engineering approach involves components from the macroscopic to the nanoscopic level, including appropriate 3D scaffolds, cells and growth factors. In this study, hexagonal scaffolds of different diagonals were fabricated by Direct Laser Writing using a photopolymerizable hybrid material. The proliferation of bone marrow (BM) mesenchymal stem cells (MSCs) cultured on structures with various diagonals, 50, 100, 150 and 200µm increased significantly after 10days in culture, however without significant differences among them. Next, recombinant human bone morphogenetic protein 2 (rhBMP-2) was immobilized onto the hybrid material both via covalent binding and physical adsorption. Both immobilization types exhibited similar high releaseate bioactivity profiles and a sustained delivery of rhBMP-2. The collagen and calcium levels produced in the extracellular matrix (ECM) were significantly elevated for the samples functionalized with BMP-2 compared to those in the osteogenic medium. Furthermore, significant upregulation of gene expression in both types of BMP-2 immobilized scaffolds was observed for alkaline phosphatase (ALPL) and osteocalcin (BGLAP) at days 7, 14, and 21, for RUNX2 at day 21, and for osteonectin (SPARC) at days 7 and 14. The results suggest that the release of bioactive rhBMP-2 from the hybrid scaffolds enhance the control over the osteogenic differentiation during cell culture.

Endothelial progenitor cells as markers of severity in hypertrophic cardiomyopathy.

AIMS: Endothelial progenitor cells (EPCs) are bone marrow-derived cells that are mobilized into the circulation to migrate and differentiate into mature endothelial cells contributing to post-natal physiological and pathological neovascularization. In this study, we evaluated circulating EPCs in patients with hypertrophic cardiomyopathy (HCM) and examined a potential association with clinical parameters of the disease. METHODS AND RESULTS: We included 40 HCM patients and 23 healthy individuals. Using flow cytometry we measured EPCs in peripheral blood as two subpopulations of CD45-/CD34+/VEGFR2+ and CD45-/CD34+/CD133+ cells. Circulating CD45-/CD34+/VEGFR2+ cells were significantly increased in HCM patients in comparison with the controls (0.000238 ± 0.0003136 vs. 0.000057 ± 0.0001316, respectively, P = 0.002). However, there was no significant difference in the number of circulating CD45-/CD34+/CD133+ cells (0.003079 ± 0.0033288 vs. 0.002065 ± 0.0022173, respectively, P = 0.153). The CD45-/CD34+/VEGFR2+ subpopulation revealed a moderate correlation with LV mass index (r = 0.35, P = 0.026), while both EPC subpopulation levels showed strong positive correlations with th E/e' ratio (r = 0.423, P = 0.007 for CD45-/CD34+/VEGFR2+ and r = 0.572, P < 0.001 for CD45-/CD34+/CD133+). CONCLUSION: HCM patients showed an increased mobilization of EPCs compared with healthy individuals that correlated with diastolic dysfunction. Our findings may open up new dimensions in the pathophysiology, prognostication, and treatment of HCM.

Monitoring interhospital transfers in Western Greece during 2003-2011: its role in health policy.

INTRODUCTION: Interhospital transfers (ITs) could provide insight into regional healthcare efficiency and evidence for policy-making. The aim of this study was to analyse ITs carried out in the Western Greece region over a nine-year period. METHODS: Archives of the National Center of Emergency Medical Services of Patras and official healthcare resources were used to analyze patient transfers from rural to 'reception' hospitals in the area, during the period 2003-2011, by hospital, medical, seasonal and population variations. RESULTS: A total of 2500 ITs from the eight rural hospitals to the central ones in the metropolitan area of Patras were monitored yearly. Transfer rates per population ranged between less than 0.3% and more than 1.0%. Only a few patients transferred outside the area (0.9%). Almost 10% of total transfers regarded diagnostic evaluation (mostly CT scan). Transfer rates were inversely related to hospital admission rates (Pearson -0.973, p=0.027), while time (in minutes) (Pearson -0.903, =0.036) and distance (in kilometers) between the rural and central hospitals (Pearson -0.907, p=0.034) also exhibited significant relationships. The level of understaffing does not have a clear effect on ITs. CONCLUSIONS: By monitoring ITs, it becomes evident where efforts should be prioritized and which of the interconnections should be optimized in a specific network of health care. In this case, interventions should be focused towards the (a) very high transfer rates from the general hospital (GH) of Aigio, (b) lack of orthopedists at GH Kalavryta, which could provide a 24 hour emergency service in a tourist ski area, (c) understaffing in the microbiological laboratory and lack of a CT scanner at GH Mesologi, and (d) lack of radiologists in several hospitals, rendering the installed equipment worthless. By monitoring the ITs, real needs and win-win actions may emerge in the complex interplay of infrastructural factors.

Circulating mesenchymal stem cells in patients with hypertrophic cardiomyopathy.

BACKGROUND: This study examines the mobilization of mesenchymal stem cells (MSCs) in patients with hypertrophic cardiomyopathy (HCM) compared to healthy individuals. The pathogenesis of myocardial hypertrophy in HCM is not fully understood. MSCs are involved in the process of neovascularization, fibrosis, and ventricular wall remodeling. METHODS AND RESULTS: We included 40 patients with HCM and 23 healthy individuals. Using flow cytometry, we measured MSCs in peripheral blood, as a population of CD45-/CD34-/CD90+ cells and also as a population of CD45-/CD34-/CD105+ cells. The resulting MSC counts were expressed as percentages of the total cells. Patients with HCM were found to have a greater percentage of circulating CD45-/CD34-CD34-/CD90+ cells compared to controls (0.0041±0.005% vs. 0.0007±0.001%, respectively, P<.001). No significant difference in circulating CD45-/CD34-/CD105+ cells in the peripheral blood was found between HCM patients and controls (0.016±0.018% vs. 0.012±0.014%, respectively, P=.4). Notably, circulating CD45-/CD34-/CD90+ cells were positively correlated with left ventricular mass index (r=0.54, P<.001). CONCLUSIONS: Patients with HCM reveal an increased mobilization of MSCs compared to healthy individuals. Although further research is needed to reveal the clinical significance of our findings, our data open a new dimension in the pathophysiology of the disease and may indicate new future therapeutic possibilities.

Increased mobilization of mesenchymal stem cells in patients with essential hypertension: the effect of left ventricular hypertrophy.

Stem cells have great clinical significance in many cardiovascular diseases. However, there are limited data regarding the involvement of mesenchymal stem cells (MSCs) in the pathophysiology of arterial hypertension. The aim of this study was to investigate the circulation of MSCs in patients with essential hypertension. The authors included 24 patients with untreated essential hypertension and 19 healthy individuals. Using flow cytometry, MSCs in peripheral blood, as a population of CD45-/CD34-/CD90+ cells and also as a population of CD45-/CD34-/CD105+ cells, were measured. The resulting counts were translated into the percentage of MSCs in the total cells. Hypertensive patients were shown to have increased circulating CD45-/CD34-/CD90+ compared with controls (0.0069%±0.012% compared with 0.00085%±0.0015%, respectively; P=.039). No significant difference in circulating CD45-/CD34-/CD105+ cells was found between hypertensive patients' and normotensive patients' peripheral blood (0.018%±0.013% compared with 0.015%±0.014%, respectively; P=.53). Notably, CD45-/CD34-/CD90+ circulating cells were positively correlated with left ventricular mass index (LVMI) (r=0.516, P<.001). Patients with essential hypertension have increased circulating MSCs compared with normotensive patients, and the number of MSCs is correlated with LVMI. These findings contribute to the understanding of the pathophysiology of hypertension and might suggest a future therapeutic target.

Biochemical characterisation of Troponin C mutations causing hypertrophic and dilated cardiomyopathies.

Cardiac muscle contraction occurs through an interaction of the myosin head with the actin filaments, a process which is regulated by the troponin complex together with tropomyosin and is Ca(2+) dependent. Mutations in genes encoding sarcomeric proteins are a common cause of familial hypertrophic and dilated cardiomyopathies. The scope of this review is to gather information from studies regarding the in vitro characterisation of six HCM and six DCM mutations on the cardiac TnC gene and to suggest, if possible, how they may lead to dysfunction. Since TnC is the subunit responsible for Ca(2+) binding, mutations in the TnC could possibly have a strong impact on Ca(2+) binding affinities. Furthermore, the interactions of mutant TnCs with their binding partners could be altered. From the characterisation studies available to date, we can conclude that the HCM mutations on TnC increase significantly the Ca(2+) sensitivity of force development or of ATPase activity, producing large pCa shifts in comparison to WT TnC. In contrast, the DCM mutations on TnC have a tendency to decrease the Ca(2+) sensitivity of force development or of ATPase activity in comparison to WT TnC. Furthermore, the DCM mutants of TnC are not responsive to the TnI phosphorylation signal resulting in filaments that preserve their Ca(2+) sensitivity in contrast to WT filaments that experience a decrease in Ca(2+) sensitivity upon TnI phosphorylation.

Circulating endothelial progenitor cells in hypertensive patients with increased arterial stiffness.

The potential association between arterial stiffening and circulating endothelial progenitor cells (EPCs) in patients with essential hypertension was investigated. Pulse wave velocity (PWV) was used to evaluate arterial stiffness in 24 patients with essential hypertension and 19 healthy controls. Blood samples were taken and immunostained with antibodies against the cell surface markers CD34, CD45, and CD133. Using flow cytometry, EPCs as a population of CD45-/CD34+/CD133+ cells were measured. Hypertensive patients were not found to have higher levels of circulating CD45-/CD34+/CD133+ compared with the control group (0.0026%±0.0031% vs 0.0023%±0.0023%, respectively; P=.7). Correlation analysis revealed a strong association between the number of CD45-/CD34+/CD133+ cells and PWV (r=0.58, P<.001), indicating that hypertensive patients with increased PWV have a greater percentage of CD45-/CD34+/CD133+ cells. Data showed a correlation between the number of circulating CD45-/CD34+/CD133+ cells and arterial stiffness, suggesting that those cells might have a role in arterial remodeling.

α-Tropomyosin with a D175N or E180G mutation in only one chain differs from tropomyosin with mutations in both chains.

α-Tropomyosin (Tm) carrying hypertrophic cardiomyopathy mutation D175N or E180G was expressed in Escherichia coli. We have assembled dimers of two polypeptide chains in vitro that carry one (αα*) or two (α*α*) copies of the mutation. We found that the presence of the mutation has little effect on dimer assembly, thereby predicting that individuals heterozygous for the Tm mutations are likely to express both αα* and α*α* Tm. Depending on the expression level, the heterodimer may be the predominant form in individuals carrying the mutation. Thus, it is important to define differences in the properties of Tm molecules carrying one or two copies of the mutation. We examined the Tm homo- and heterodimer properties: actin affinity, thermal stability, calcium regulation of myosin subfragment 1 binding, and calcium regulation of myofibril force. We report that the properties of the heterodimer may be similar to those of the wild-type homodimer (actin affinity, thermal stability, D175N αα*), similar to those of the mutant homodimer (calcium sensitivity, D175N αα*), intermediate between the two (actin affinity, E180G αα*), or different from both (thermal stability, E180G αα*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.

In vitro formation and characterization of the skeletal muscle α·ß tropomyosin heterodimers.

Tropomyosin (Tm) is a dimer made of two alpha helical chains associated into a parallel coiled-coil. In mammalian skeletal and cardiac muscle, the Tm is expressed from two separate genes to give the α- and ß-Tm isoforms. These associate in vivo to form homo- (α(2)) and heterodimers (α·ß) with little ß(2) normally observed. The proportion of α(2) vs α·ß varies across species and across muscle types from almost 100% α(2)- to 50% α·ß-Tm. The ratio can also vary during development and in disease. The functional significance of the presence of these two isoforms has not been defined because it is difficult to isolate or purify the α·ß dimer for functional studies. Here we report an effective method for purifying bacterially expressed Tm as α·ß dimers using a cleavable N-terminal tag on one of the two chains. The same method can be used to isolate Tm dimers in which one chain carries a mutation. We go on to show that the α·ß dimers differ in key properties (actin affinity, thermal stability) from either the α(2)- or ß(2)-Tm. However, the ability to regulate myosin binding when combined with cardiac troponin appears unaffected.