Khon C. Huynh

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.

Shear-related fibrillogenesis of fibronectin.

Abstract Biomechanical forces can induce the transformation of fibronectin (Fn) from its compact structure to an extended fibrillar state. Adsorption of plasma proteins onto metallic surfaces may also influence their conformation. We used a cone-plate rheometer to investigate the effect of shear and stainless steel on conformational changes of Fn. In control experiments, cones grafted once or twice with polyethylene glycol were used. Plasma Fn was added at concentrations of 50 or 100 μg/ml to bovine serum albumin (BSA)- or Fn-coated plates and subsequently exposed to dynamic shear rates stepwise increasing from 50 to 5000 s-1 within 5 min and subsequently decreasing from 5000 to 50 s-1 within 5 min. The viscosity (mPa s) of Fn solutions was recorded over 10 min. Upon exposure to shear, the viscosity in the sample increased, suggesting conformational changes in Fn. Western blotting and densitometric analyses demonstrated that conformational changes of plasma Fn depended both on shear and protein concentration. However, there was no significant difference in fibril formation between BSA- or Fn-coated plates, suggesting that physical properties of stainless steel and biomechanical forces such as shear can affect the molecular structure of Fn. Our model may provide useful information of surface- and flow-induced alterations of plasma proteins.

Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis

Fibronectin (FN), a dimeric adhesive glycoprotein, which is present both in plasma and the extracellular matrix can interact with platelets and thus contribute to platelet adhesion and aggregation. It has been shown that FN can decrease platelet aggregation but enhance platelet adhesion, suggesting a dual role of FN in haemostasis. The prevalent function(s) of FN may be determined by its fibril form. To explore the suggested dual role of this adhesive protein for haemostasis in further detail, we now tested for any differences of adherent and suspended platelets with regard to their effect to unfold and assemble FN upon interaction. Platelet aggregation and adhesion assays were performed using washed platelets in the presence of exogenous FN. Addition of plasma FN reduced platelet aggregation in response to collagen or PMA by 50% or 25% but enhanced platelet adhesion onto immobilized collagen, as compared to control experiments. Analyses by fluorescence resonance energy transfer (FRET) demonstrated that adherent platelets but not suspended platelets were capable of unfolding FN during 3h incubation. Fluorescence microscopy and deoxycholate (DOC) solubility assays demonstrated that FN fibrils formed only on the surfaces of adherent platelets. In addition, platelets adherent onto FN revealed a significantly higher activity of specific Src phosphorylation (pY418) than platelets in suspension. These data suggest (1) that the function of FN in haemostasis is prevalent to its assembly, unfolding and subsequent fibril formation on the surface of adherent platelets and (2) that outside-in signaling contributes to the interaction of platelets and FN.

Improvement of the in vivo cellular repopulation of decellularized cardiovascular tissues by a detergent-free, non-proteolytic, actin-disassembling regimen: Biological decellularization of cardiovascular implants

Low immunogenicity and high repopulation capacity are crucial determinants for the functional and structural performance of acellular cardiovascular implants. The present study evaluates a detergent‐free, non‐proteolytic, actin‐disassembling regimen (BIO) for decellularization of heart valve and vessel grafts, particularly focusing on their bio‐functionality. Rat aortic conduits (rAoC; n = 89) and porcine aortic valve samples (n = 106) are decellularized using detergents (group DET) or the BIO regimen. BIO decellularization results in effective elimination of cellular proteins and significantly improves removal of DNA as compared with group DET, while the extracellular matrix (ECM) structure as well as mechanical properties are preserved. The architecture of rAoC in group BIO allows for improved bio‐functionalization with fibronectin (FN) in a standardized rat implantation model: BIO treatment significantly increases speed and amount of autologous medial cellular repopulation in vivo (p < 0.001) and decreases the formation of hyperplastic intima (p < 0.001) as compared with FN‐coated DET‐decellularized grafts. Moreover, there are no signs of infiltration with inflammatory cells. The present biological, detergent‐free, non‐proteolytic regimen balances effective decellularization and ECM preservation in cardiovascular grafts, and provides optimized bio‐functionality. Additionally, this study implies that the actin‐disassembling regimen may be a promising approach for bioengineering of acellular scaffolds from other muscular tissues, as for example myocardium or intestine. Copyright

Investigate the Effect of Thawing Process on the Self-Assembly of Silk Protein for Tissue Applications

Biological self-assembly is a process in which building blocks autonomously organize to form stable supermolecules of higher order and complexity through domination of weak, noncovalent interactions. For silk protein, the effect of high incubating temperature on the induction of secondary structure and self-assembly was well investigated. However, the effect of freezing and thawing on silk solution has not been studied. The present work aimed to investigate a new all-aqueous process to form 3D porous silk fibroin matrices using a freezing-assisted self-assembly method. This study proposes an experimental investigation and optimization of environmental parameters for the self-assembly process such as freezing temperature, thawing process, and concentration of silk solution. The optical images demonstrated the possibility and potential of −80ST48 treatment to initialize the self-assembly of silk fibroin as well as controllably fabricate a porous scaffold. Moreover, the micrograph images illustrate the assembly of silk protein chain in 7 days under the treatment of −80ST48 process. The surface morphology characterization proved that this method could control the pore size of porous scaffolds by control of the concentration of silk solution. The animal test showed the support of silk scaffold for cell adhesion and proliferation, as well as the cell migration process in the 3D implantable scaffold.

Impact of shear stress on Src and focal adhesion kinase phosphorylation in fibrinogen-adherent platelets.

&NA; Shear stress alone can activate platelets resulting in a subsequent platelet aggregation, so-called ‘shear-induced platelet aggregation’. In our work, we analyzed how differently elevated shear stress impacts the Src and focal adhesion kinase (FAK) activation in fibrinogen-adherent human platelets. We detected the extents of Src pY418 and FAK pY397 activations in platelets on immobilized fibrinogen and over BSA under shear conditions. Moreover, we analyzed the role of &agr;IIb&bgr;3 in the shear-induced platelet signaling by performing our experiments in the presence of the &agr;IIb&bgr;3-antagonist Abciximab. Abnormally high shear rates (5000 s−1) significantly increased the extent of phosphorylation of both tyrosine kinases after short (2 min) incubation time independently of the presence or absence of the integrin &agr;IIb&bgr;3 ligand, fibrinogen. We could see considerably greater Src activation on immobilized fibrinogen than on BSA, but the extent of FAK Y397 phosphorylation was independent on the matrix. Abciximab not only reduced the Src and FAK signaling in platelets exposed to 5000 s−1 on immobilized fibrinogen, but in platelets exposed to 5000 s−1 over BSA as well. Our data indicate that whereas Src activation under shear stress is dominantly ligand-dependent, FAK signaling seems to be mostly shear induced.

Contribution of distinct platelet integrins to binding, unfolding, and assembly of fibronectin.

Abstract Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbβ3, αvβ3, and α5β1 are known to assemble FN fibrils on platelets. In our study, we examined the contribution of these integrins to FN binding, unfolding, and assembly on platelets in suspension and adherent platelets in the presence or absence of agonists. Phorbol 12-myristate 13-acetate (PMA), but not adenosine diphosphate (ADP), induced binding of FN to platelets in suspension. In contrast, adherent platelets were able to deposit FN on their surfaces in the absence of agonists. β3 integrins had a major impact on the interaction of FN on platelets. αvβ3 showed a similar contribution to the binding of FN as αIIbβ3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5β1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than β3 integrins. None of the distinct antibodies directed against one of the three integrins caused a complete inhibition of binding, unfolding, and assembly of FN by platelets. Thus, it is likely that αIIbβ3, αvβ3, and α5β1 or another still unknown receptor can be substituted.

Integrin αIIbβ3-Dependent ERK Signaling Is Regulated by Src and Rho Kinases in Both Leu33 and Pro33 Polymorphic Isoforms

Platelet integrin αIIbβ3 possesses a Leu/Pro polymorphism at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b). The Pro33 isoform has been suggested to exhibit prothrombotic features. αIIbβ3-expressing CHO (Chinese hamster ovary) cells on immobilized fibrinogen show activation of the MAP kinase family member ERK2, with an enhanced ERK2 activity in Pro33 cells compared to Leu33 cells. In our present work, we examined how the Leu/Pro polymorphism modulates the ERK2 activation stimulated by 2 differently triggered outside-in signalings. We either treated the CHO cells with Mn2+ or allowed them to adhere to fibrinogen. Moreover, we studied which signaling cascades are involved in ERK2 activation. In contrast to immobilized fibrinogen, Mn2+ did not significantly increase ERK2 activation. However, Mn2+ had a synergistic effect on ERK2 phosphorylation when combined with immobilized fibrinogen. Pro33 cells adherent to fibrinogen exhibited a significantly greater ERK2 activity than Leu33 cells in the presence of Mn2+, which peaked after 10 min of adhesion. Our data showed that Src family and rho kinases play a crucial role in the integrin αIIbβ3-dependent outside-in signaling to ERK2.

Characterization of Fibronectin Assembly by Adherent Platelets Under Flow Conditions: Effect of Shear Stress and Role of β3 Integrins

Platelet thrombi at site of vascular injury require resistance to shear stress generated by the flowing blood. Fibronectin assembly can contribute to platelet adhesion and aggregation. Here, we examined the effect of shear stress and the role of αIIbβ3 and αvβ3 in fibronectin assembly by adherent platelets under flow dynamic conditions in vitro. Platelets were placed onto immobilized fibronectin (50 µg/ml) with fluorescently labeled fibronectin. Shear rates (500 s−1 or 5000 s−1) were generated by a viscometer (Diamed) for 2 or 10 min. Adherent platelets were lysed with 2% deoxycholate buffer. Insoluble fibronectin fibrils were isolated by centrifugation for quantification by measuring fluorescence intensity. In some experiments, abciximab (anti-β3) or LM609 (anti-αvβ3) antibody, were added before experiments. When shear rates increased from 500 to 5000 s−1, the amount of insoluble fibronectin detectable on platelets increased significantly (p < 0.05). However, prolongation of the exposure time to shear did not cause a significant difference in fibronectin assembly. No fibronectin fibrils were detectable on adherent platelets under static conditions. After 2 min at 5000 s−1, platelets blocked with abciximab showed a significant decrease in fibril formation in comparison to control experiments (no antibody). A similar inhibitory effect was achieved with LM609. When platelets were exposed to 5000 s−1 for 10 min, abciximab showed a higher inhibition on fibronectin assembly than LM609. We document that fibronectin assembly by adherent platelets is strongly affected by flow conditions. αvβ3 provides a significant contribution in initial phase, while αIIbβ3 probably exerts its effect in the later phase of fibronectin assembly under high shear conditions.