Payam Akhyari

Myocardial tissue engineering: the extracellular matrix

More than a decade after the first reports on successful three-dimensional cardiac cell culture for experimental and potential therapeutic application, the interest and experimental efforts in the field of myocardial tissue engineering continues to grow. The hope that tissue cultures may one day act as graft substitute for malfunctioning myocardium continues to drive current scientific activity. Against this background interest seem to have progressively shifted towards the aim of engineering single tissue components. Accordingly, elements of the extracellular matrix (ECM) have gained increasing attention as potentially crucial mediators in developing and maintaining the characteristics of three-dimensional cardiac cell cultures. The ECM is now no longer regarded as merely a scaffold for developing tissue, a concept that is widely acknowledged in modern tissue engineering. The understanding of the role of precursor and stem cells has highlighted new complicated aspects of cell proliferation and differentiation and ECM proves to play an important role in providing essential signals to influence major intracellular pathways such as proliferation, differentiation and cell metabolism. Furthermore, progress in biochemical engineering has provided the perspective of application of synthetic ECM-linked molecules with bioactive potential. With the advent and continuous refinement of cell removal techniques, a new class of native acellular ECM has emerged with some striking advantages. The presently available ECM materials aim to closely resemble the in vivo microenvironment by acting as an active component of the developing tissue construct. It is therefore not surprising that most of the focus in myocardial tissue engineering has been on cell-matrix interaction, for both naturally derived and synthetic ECM. This article provides a review of established models of myocardial tissue engineering with respect to the employed ECM materials including current frontiers in material development.

Sternal microcirculation after skeletonized versus pedicled harvesting of the internal thoracic artery: a randomized study.

OBJECTIVE In human patients the influence of skeletonized internal thoracic artery harvesting on the sternal microcirculation in the perioperative phase has not been well investigated. METHODS Twenty-four consecutive male patients who were scheduled for isolated coronary artery bypass grafting were prospectively randomized into 2 groups. The left internal thoracic artery was harvested by using the skeletonized technique in group 1, and it was harvested with a pedicle in group 2. Superficial (2 mm) and deep (8 mm) tissue oxygen saturation and blood flow were measured presternally and retrosternally in the upper, middle, and lower sternal parts with a novel laser Doppler flowmetric and remission spectroscopic system (Oxygen-to-See; LEA Medizintechnik, Giessen, Germany). RESULTS Presternal tissue oxygen saturation deteriorated at the upper and middle sternum, and presternal blood flow deteriorated at all measurement points after internal thoracic artery harvesting in both groups. Skeletonization had no advantage in maintaining presternal microcirculation. Retrosternal microcirculation also deteriorated at all measurement points after internal thoracic artery harvesting in both groups. However, the deterioration of the retrosternal microcirculation was significantly less in group 1 at the middle and lower sternum; values of oxygen saturation to the baseline were 86% +/- 3.8% versus 60% +/- 4.3% (P = .001) at 2-mm depth and 82% +/- 4.2% versus 61% +/- 6.1% (P = .009) at 8-mm depth at the middle sternum and 95% +/- 3.2% versus 78% +/- 1.3% (P = .001) at 2-mm depth and 94% +/- 2.2% versus 78% +/- 4.6% (P = .004) at 8-mm depth at the lower sternum in groups 1 and 2, respectively. CONCLUSION The damage of the tissue microcirculation in the middle and lower retrosternal area is significantly less after internal thoracic artery skeletonization compared with that after the pedicled internal thoracic artery harvesting technique.

Secretory Products From Epicardial Adipose Tissue of Patients With Type 2 Diabetes Mellitus Induce Cardiomyocyte Dysfunction

Background— Secreted factors from epicardial adipose tissue (EAT) have been implicated in the development of cardiomyocyte dysfunction. This study aimed to assess whether alterations in the secretory profile of EAT in patients with type 2 diabetes mellitus (DM2) affect contractile function and insulin action in cardiomyocytes. Methods and Results— Contractile function and insulin action were analyzed in primary adult rat cardiomyocytes incubated with conditioned media (CM) generated from explants of EAT biopsies obtained from patients without and with DM2. CM from subcutaneous and pericardial adipose tissue biopsies from the same patients served as the control. Cardiomyocytes treated with CM (EAT) from DM2 patients showed reductions in sarcomere shortening, cytosolic Ca2+ fluxes, expression of sarcoplasmic endoplasmic reticulum ATPase 2a, and decreased insulin-mediated Akt-Ser473-phosphorylation as compared with CM from the other groups. Profiling of the CM showed that activin A, angiopoietin-2, and CD14 selectively accumulated in CM-EAT-DM2 versus CM-EAT in patients without DM2 and CM from the other fat depots. Accordingly, EAT biopsies from DM2 patients were characterized by clusters of CD14-positive monocytes. Furthermore, SMAD2-phosphorylation, a downstream target of activin A signaling, was elevated in cardiomyocytes treated with CM (EAT) from DM2 patients, and the detrimental effects of CM (EAT) from DM2 patients were partially abolished in cardiomyocytes pretreated with a neutralizing antibody against activin A. Finally, both recombinant activin A and angiopoietin-2 reduced cardiomyocyte contractile function, but only activin A reduced the expression of sarcoplasmic endoplasmic reticulum ATPase 2a. Conclusions— Collectively, our data implicate DM2-related alterations in the secretory profile of EAT in the pathogenesis of diabetes mellitus–related heart disease.

Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating

Decellularization is a promising option to diminish immune and inflammatory response against donor grafts. In order to accelerate the autologous in vivo recellularization of aortic conduits for an enhanced biocompatibility, we tested fibronectin surface coating in a standardized rat implantation model. Detergent-decellularized rat aortic conduits (n = 36) were surface-coated with covalently Alexa488-labeled fibronectin (50 μg/ml, 24 h) and implanted into the systemic circulation of Wistar rats for up to 8 weeks (group FN; n = 18). Uncoated implants served as controls (group C; n = 18). Fibronectin-bound fluorescence on both surfaces of the aortic conduits was persistent for at least 8 weeks. Cellular repopulation was examined by histology and immunofluorescence (n = 24). Luminal endothelialization was significantly accelerated in group FN (p = 0.006 after 8 weeks), however, local myofibroblast hyperplasia with significantly increased ratio of intima-to-media thickness occurred (p = 0.0002 after 8 weeks). Originating from the adventitial surface, alpha-smooth muscle actin and desmin positive cell invasion into the media of fibronectin-coated conduits was significantly increased as compared to group C (p < 0.0001). In these medial areas, in situ zymography revealed enhanced matrix metalloproteinase activity. In both groups, inflammatory cell markers (CD3 and CD68) and signs of thrombosis proved negative. With regard to several markers of cell adhesion, inflammation and calcification, quantitative real-time PCR (n = 12) revealed no significant inter-group differences. Fibronectin surface coating of decellularized cardiovascular implants proved feasible and persistent for at least 8 weeks in the systemic circulation. Biofunctional protein coating accelerated the autologous in vivo endothelialization and induced a significantly increased medial recellularization. Therefore, this strategy may contribute to the improvement of current clinically applied bioprostheses.

Cardioprotective properties of omentin-1 in type 2 diabetes: evidence from clinical and in vitro studies.

Context Adipokines are linked to the development of cardiovascular dysfunction in type 2 diabetes (DM2). In DM2-patients, circulating levels of omentin-1, an adipokine preferentially expressed in epicardial adipose tissue, are decreased. This study investigated whether omentin-1 has a cardioprotective function. Methods Omentin-1 levels in plasma and cardiac fat depots were determined in DM2-patients versus controls. Moreover, the relation between omentin-1 levels and cardiac function was examined in men with uncomplicated DM2. Finally, we determined whether omentin-1 could reverse the induction of cardiomyocyte dysfunction by conditioned media derived from epicardial adipose tissue from patients with DM2. Results Omentin-1 was highly expressed and secreted by epicardial adipose tissue, and reduced in DM2. Circulating omentin-1 levels were lower in DM2 versus controls, and positively correlated with the diastolic parameters early peak filling rate, early deceleration peak and early deceleration mean (all P<0.05). The improved diastolic function following pioglitazone treatment associated with increases in omentin-1 levels (P<0.05). In vitro, exposure of cardiomyocytes to conditioned media derived from epicardial adipose tissue from patients with DM2 induced contractile dysfunction and insulin resistance, which was prevented by the addition of recombinant omentin. Conclusion These data identify omentin-1 as a cardioprotective adipokine, and indicate that decreases in omentin-1 levels could contribute to the induction of cardiovascular dysfunction in DM2.

Transplantation material bovine pericardium: biomechanical and immunogenic characteristics after decellularization vs. glutaraldehyde-fixing

Hülsmann J, Grün K, El Amouri S, Barth M, Hornung K, Holzfuß C, Lichtenberg A, Akhyari P. Transplantation material bovine pericardium: biomechanical and immunogenic characteristics after decellularization vs. glutaraldehyde‐fixing. Xenotransplantation 2012; 19: 286–297.

Clinically Established Hemostatic Scaffold (Tissue Fleece) as Biomatrix in Tissue- and Organ-Engineering Research

Various types of three-dimensional matrices have been used as basic scaffolds in myocardial tissue engineering. Many of those are limited by insufficient mechanical function, availability, or biocompatibility. We present a clinically established collagen scaffold for the development of bioartificial myocardial tissue. Neonatal rat cardiomyocytes were seeded into Tissue Fleece (Baxter Deutschland, Heidelberg, Germany). Histological and ultrastructural examinations were performed by DAPI and DiOC(18) staining and electron microscopy, respectively. Force measurements from the spontaneously beating construct were obtained. The constructs were stimulated with agents such as adrenalin and calcium, and by stretching. Passive stretch curves were obtained. Spontaneous contractions of solid bioartificial myocardial tissue (BMT), 20 x 15 x 2 mm, resulted. Contractions continued to week 12 (40% of BMTs) in culture. Histology revealed intercellular and also cell-fibril junctions. Elasticity was similar to that of native rat myocardium. Contractile force increased after topical administration of Ca(2+) and adrenaline. Stretch led to the highest levels of contractile force. In summary, bioartificial myocardial tissue with significant in vitro longevity, spontaneous contractility, and homogeneous cell distribution was produced using Tissue Fleece. Tissue Fleece constitutes an effective scaffold to engineer solid organ structures, which could be used for repair of congenital defects or replacement of diseased tissue.

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.

Stem cells used for cardiovascular tissue engineering

Stem cell research and tissue engineering have become leading fields in basic research worldwide. Especially in cardiovascular medicine, initial reports on the potential of using stem cells to recover cardiac function and replace organ subunits such as heart valves seemed to offer the promise of widespread clinical use in the near future. However, the broad application of this new therapy failed due to safety and efficacy concerns. Due in part to the initial reports, major basic research efforts were undertaken to explore the specific cell types in greater detail and identify their mechanisms of supporting function, resulting in remarkable new findings in stem cell biology. For example, the notion of resident human cardiac stem cells has disproved the earlier supposition that the human heart is a finitely differentiated organ without the intrinsic potential for regeneration. Furthermore, new technologies emerged to produce pluripotent cells without the ethical and immunological drawbacks of embryonic stem cells (for instance by nuclear transfer). Other autologous cell sources are presently under investigation in myocardial tissue engineering. For tissue engineering of heart valves and small calibre vessels, the use of autologous endothelial (precursor) cells may be the optimal means of seeding a biological or artificial scaffold. It is important that ongoing basic and clinical research in cardiovascular surgery might explore the potential of different cell types either using tissue engineering constructs or in cell transplantation approaches.

Activin A impairs insulin action in cardiomyocytes via up-regulation of miR-143

AIMS Enhanced activin A release from epicardial adipose tissue (EAT) has been linked to the development of cardiac dysfunction in type 2 diabetes (T2D). This study examined whether the inhibition of insulin action induced by epicardial adipokines in cardiomyocytes can be ascribed to alterations in miRNA expression. METHODS AND RESULTS Expression levels of miRNAs were assessed by real-time PCR in primary adult rat cardiomyocytes (ARC) exposed to conditioned media generated from EAT biopsies (CM-EAT) from patients with and without T2D. CM-EAT-T2D altered the expression of eight miRNAs in ARC vs. CM-EAT from patients without T2D. Of these, only expression of the miR-143/145 cluster was affected by activin A in the same direction as CM-EAT-T2D. Accordingly, activin A neutralizing antibodies prevented the induction of the miR-143/145 cluster by CM-EAT-T2D. Subsequently, the impact of the miR-143/145 cluster on insulin action was investigated. Transfection of HL-1 cells with precursor-miR-143 (pre-miR-143), but not pre-miR-145, blunted the insulin-mediated phosphorylation of Akt and its substrate proline-rich Akt substrate of 40 kDa (PRAS40), and reduced insulin-stimulated glucose uptake. Also lentivirus-mediated expression of pre-miR-143 in ARC reduced insulin-induced Akt phosphorylation. These effects were ascribed to down-regulation of the miR-143 target and regulator of insulin action, the oxysterol-binding protein-related protein 8 (ORP8) in both ARC and HL-1 cells. Finally, LNA-anti-miR-143 protected against the detrimental effects of CM-EAT-T2D on insulin action in ARC. CONCLUSION Activin A released from EAT-T2D inhibits insulin action via the induction of miR-143 in cardiomyocytes. This miRNA inhibits the Akt pathway through down-regulation of the novel regulator of insulin action, ORP8.