An altered pattern of myocardial histopathological and molecular changes underlies the different characteristics of type-1 and type-2 diabetic cardiac dysfunction.

Increasing evidence suggests that both types of diabetes mellitus (DM) lead to cardiac structural and functional changes. In this study we investigated and compared functional characteristics and underlying subcellular pathological features in rat models of type-1 and type-2 diabetic cardiomyopathy. Type-1 DM was induced by streptozotocin. For type-2 DM, Zucker Diabetic Fatty (ZDF) rats were used. Left ventricular pressure-volume analysis was performed to assess cardiac function. Myocardial nitrotyrosine immunohistochemistry, TUNEL assay, hematoxylin-eosin, and Masson's trichrome staining were performed. mRNA and protein expression were quantified by qRT-PCR and Western blot. Marked systolic dysfunction in type-1 DM was associated with severe nitrooxidative stress, apoptosis, and fibrosis. These pathological features were less pronounced or absent, while cardiomyocyte hypertrophy was comparable in type-2 DM, which was associated with unaltered systolic function and increased diastolic stiffness. mRNA-expression of hypertrophy markers c-fos, c-jun, and ß-MHC, as well as pro-apoptotic caspase-12, was elevated in type-1, while it remained unaltered or only slightly increased in type-2 DM. Expression of the profibrotic TGF-ß 1 was upregulated in type-1 and showed a decrease in type-2 DM. We compared type-1 and type-2 diabetic cardiomyopathy in standard rat models and described an altered pattern of key pathophysiological features in the diabetic heart and corresponding functional consequences.

A novel culture device for the evaluation of three-dimensional extracellular matrix materials.

Cell-matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell-matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell-ECM interactions in 3D ECM.

The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities.

Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macroarchitecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning the microcomposition of the ECM scaffolds on a histological and biochemical level. While laminin could still be detected in all groups, other crucial ECM components, like elastin and collagen IV, were completely removed by at least one of the protocols. Further, only three protocols maintained a glycosaminoglycan content comparable to native tissue, whereas the remaining DNA content within the ECM varied highly throughout all four tested protocols. This study showed that the degree of acellularity and resulting ECM composition of decellularized myocardial scaffolds strongly differs depending on the decellularization protocol.

Opposite effects of transforming growth factor-ß1 and vascular endothelial growth factor on the degeneration of aortic valvular interstitial cell are modified by the extracellular matrix protein fibronectin: implications for heart valve engineering.

The enhancement of valvular interstitial cell (VIC) calcification by transforming growth factor-ß1 (TGF-ß1) and the endothelial inducing effect of vascular endothelial growth factor (VEGF) have been demonstrated. Here we report the modulating properties of extracellular matrix (ECM) modification on VIC calcification in the presence of TGF-ß1 and VEGF. Ovine aortic VICs cultured on collagen, fibronectin, laminin, or uncoated surfaces were exposed to TGF-ß1, VEGF, or left untreated. VEGF significantly inhibited the formation of calcific nodules independent of ECM Protein coating (p < 0.05). TGF-ß1 exposition resulted in the formation of calcific nodules on collagen, laminin, and uncoated control surfaces. In contrast, fibronectin coating resulted in significantly reduced nodule formation despite TGF-ß1 administration. Further, we showed a marked increase of apoptotic and dead cells in calcific nodules. Overall, our data demonstrate that, an additive protective effect on VICs can be achieved by providing specific growth factors or a specific ECM environment. Here, VEGF administration inhibited calcification and apoptosis, particularly in combination with fibronectin coating. This combination appears to be a promising tool for modification of heart valve scaffolds for tissue engineering purposes and preclinical trials.

In vivo functional performance and structural maturation of decellularised allogenic aortic valves in the subcoronary position.

OBJECTIVE: Successful animal and clinical implantation of decellularised heart valves has been performed in the pulmonary position. Comparable results have not yet been achieved for the aortic position with the high haemodynamic demands of the systemic circulation and the challenging implantation procedure. METHODS: Allogenic aortic valves (n=10) were decellularised using detergents (decellularised aortic valves (dAoVs)). Five prostheses were analysed for decellularisation quality and scaffold preservation. Five valves were orthotopically implanted in juvenile sheep in a subcoronary technique. After 5 months, echocardiography, immunohistology, histology, electron microscopy and western blot (WB) were used for analysis. RESULTS: All animals survived the follow-up with increased body weight (38.8 ± 2.8kg vs 56.0 ± 2.6kg, p<0.001). After implantation, three dAoVs showed negligible and two others minor insufficiency (I), which remained unchanged at explantation. Effective orifice area increased slightly (1.1 ± 0.2cm(2) vs 1.6 ± 0.3cm(2), p=0.051). Explanted dAoVs (n=4) showed excellent macroscopy with minor soft-tissue nodules observed at the free cusp margins of only one dAoV. No valve showed any signs of thrombosis or calcification. On microscopic evaluation, the cusp architecture was preserved with an almost complete endothelial repopulation as confirmed by vimentin(+)/von Willebrand factor (vWF(+))-staining, WB of endothelial markers (eNOS/vWF) and scanning electron microscopy (SEM). Partial interstitial reseeding with vimentin(+)/alpha-smooth muscle (αsm(+))-cells was noted. Quantitative measurement of collagen-IV, collagen-I, laminin and elastin (WB) demonstrated preserved scaffold composition as compared to native tissue. CONCLUSION: The dAoVs showed excellent functional outcome at 5 months in a subcoronary model of juvenile sheep. Advanced endothelial and nascent interstitial repopulation, with preserved structural integrity under the high-shear-stress milieu of the aortic valve, encourage further long-term studies.

The effect of the dual Src/Abl kinase inhibitor AZD0530 on Philadelphia positive leukaemia cell lines.

BACKGROUND: Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells. METHODS: Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively. RESULTS: AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15. CONCLUSION: Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl.