Morphological dependent effect of cell-free formed supramolecular fibronectin on cellular activities.

Fibrillar fibronectin (FFN), an active form of fibronectin (FN), plays important roles in various cellular processes. Our goal is to investigate effect of FFN morphology on cellular behaviors. Plasma FN at two concentrations was cross-linked into FFN by dialysis against 2 M urea followed by morphological analysis under Scanning Electron Microscopy. To evaluate effect of FFN morphology, fibroblasts were cultured on FN or different FFNs. Fibroblast behaviors including adhesion, spreading, and migration were evaluated. Our data showed that FN fibrillogenesis was dependent on FN concentration. At high concentrations (0.75 mg/mL), large FFN approximately 2.167 + 0.875 µm in diameter were formed with attached nodular structures and rough surface. In contrast, smooth surface FFN fibrils with diameter of 1.886 + 0.412 µm were formed from FN at 0.25 mg/mL. Cellular assays revealed morphological dependent biological effects of different FFNs. Fibroblast separately adhered to native FN and remained spherical while on FFN, cells attached with higher quantity and showed spreading morphology. A synergistic ligand interaction of integrin α5ß1 and αvß3 was observed in cell adhering on FFN. Cell migration results showed that large FFN decreased migration rate while small FFN did not. Taken together, our data draws new attention towards controlling biological function of FN by its fibrillar structure.

Leu33Pro (PlA) polymorphism of integrin beta3 modulates platelet Src pY418 and focal adhesion kinase pY397 phosphorylation in response to abnormally high shear stress.

OBJECTIVES: Shear stress can activate platelet integrin-mediated signaling that leads to shear-induced platelet aggregation (SIPA) and eventually contribute to acute myocardial infarction. The major platelet integrin αIIbß3 is polymorphic at residue 33 [Leu33Pro (PlA) polymorphism]. The Pro33 isoform has been shown to have a prothrombotic phenotype. In this work, we studied the impact of Leu33/Pro33 polymorphism on the shear-induced integrin-mediated Src and FAK activation in platelets. METHODS: Platelets of both genotypes were placed on immobilized fibrinogen or heat activated BSA and were exposed to physiological (500/s) or abnormally high (5000/s) shear rates for 2-10 min. Platelets after exposure to shear were analysed for Src pY418 and FAK pY397 activities. RESULTS: Whereas physiological shear stress does not affect platelet signaling, abnormally high-shear stress considerably elevates Src and FAK phosphorylation in both Pro33 and Leu33 platelets. Both under static and flow conditions, Pro33 platelets exhibited a significantly higher Src and FAK activities than Leu33 platelets. Interestingly, even in the absence of the αIIbß3-fibrinogen interaction, we could detect a shear-induced integrin-mediated signaling of Src and FAK in platelets. In parallel experiments in which platelets were pretreated with abciximab, an integrin αIIbß3 antagonist, activation of both kinases by shear was inhibited. CONCLUSION: Taken together, our data indicates an important role of αIIbß3 and shows that Leu33Pro polymorphism modulates the integrin-mediated Src and FAK signaling in platelets in response to shear stress.

Shear-dependent fibrillogenesis of fibronectin: Impact of platelet integrins and actin cytoskeleton.

Soluble plasma fibronectin (Fn) with its inactive compact structure requires unfolding to assemble into active fibrils, which play a role in hemostasis and thrombosis. Fn fibril assembly involves Fn binding to cell receptors, biomechanical coupling of Fn to the cytoskeleton by integrins, exposure of self-assembly sites via contractile cell forces, and elongation of fibrils by Fn polymerization. In this report, we investigated the effect of platelet integrins and actin cytoskeleton on conformational changes of Fn induced by shear. Plasma Fn, in the presence or absence of washed platelets, was exposed to dynamic shear simulating venous or arterial flow conditions. Platelet integrins (αIIbß3, αvß3, and α5ß1) were blocked by inhibitory antibodies to determine their contribution to shear-induced Fn fibrillogenesis. To examine the role of platelet cytoskeleton in Fn fibrillogenesis induced by shear, platelets were preincubated with cytoskeleton drugs, i. e jasplakinolide to stabilize actin or cytochalasin D to inhibit actin polymerization. Microscopic analyses demonstrated that flow and resulting shear stress over a broad range of physiological and pathological rates (50-5000 s-1) could induce conformational changes of plasma Fn. In addition, the formation of Fn fibrils is modulated by platelet integrins. In this respect, ß3 integrins play a dominant role in terms of Fn fibrillogenesis induced by shear. Disruption of the actin polymerization markedly diminished Fn unfolding and assembly. These observations lead to the conclusion that Fn-integrin ß3-cytoskeleton interaction is crucial for the assembly of plasma Fn matrix under flow conditions.

The human platelet antigen-1b (Pro33) variant of αIIbß3 allosterically shifts the dynamic conformational equilibrium of this integrin toward the active state.

Integrins are heterodimeric cell-adhesion receptors comprising α and ß subunits that transmit signals allosterically in both directions across the membrane by binding to intra- and extracellular components. The human platelet antigen-1 (HPA-1) polymorphism in αIIbß3 arises from a Leu → Pro exchange at residue 33 in the genu of the ß3 subunit, resulting in Leu33 (HPA-1a) or Pro33 (HPA-1b) isoforms. Although clinical investigations have provided conflicting results, some studies have suggested that Pro33 platelets exhibit increased thrombogenicity. Under flow-dynamic conditions, the Pro33 variant displays prothrombotic properties, characterized by increased platelet adhesion, aggregate/thrombus formation, and outside-in signaling. However, the molecular events underlying this prothrombotic phenotype have remained elusive. As residue 33 is located >80 Å away from extracellular binding sites or transmembrane domains, we hypothesized that the Leu → Pro exchange allosterically shifts the dynamic conformational equilibrium of αIIbß3 toward an active state. Multiple microsecond-long, all-atom molecular dynamics simulations of the ectodomain of the Leu33 and Pro33 isoforms provided evidence that the Leu → Pro exchange weakens interdomain interactions at the genu and alters the structural dynamics of the integrin to a more unbent and splayed state. Using FRET analysis of fluorescent proteins fused with αIIbß3 in transfected HEK293 cells, we found that the Pro33 variant in its resting state displays a lower energy transfer than the Leu33 isoform. This finding indicated a larger spatial separation of the cytoplasmic tails in the Pro33 variant. Together, our results indicate that the Leu → Pro exchange allosterically shifts the dynamic conformational equilibrium of αIIbß3 to a structural state closer to the active one, promoting the fully active state and fostering the prothrombotic phenotype of Pro33 platelets.

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

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 © 2017 John Wiley & Sons, Ltd.

Integrin αIIbß3-Dependent ERK Signaling Is Regulated by Src and Rho Kinases in Both Leu33 and Pro33 Polymorphic Isoforms.

Platelet integrin αIIbß3 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. αIIbß3-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 αIIbß3-dependent outside-in signaling to ERK2.

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

: 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 αIIbß3 in the shear-induced platelet signaling by performing our experiments in the presence of the αIIbß3-antagonist Abciximab. Abnormally high shear rates (5000 s) 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 αIIbß3 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 on immobilized fibrinogen, but in platelets exposed to 5000 s 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.

Rheology of perfusates and fluid dynamical effects during whole organ decellularization: a perspective to individualize decellularization protocols for single organs.

The approach of whole organ decellularization is rapidly becoming more widespread within the tissue engineering community. Today it is well known that the effects of decellularization protocols may vary with the particular type of treated tissue. However, there are no methods known to individualize decellularization protocols while automatically ensuring a standard level of quality to minimize adverse effects on the resulting extracellular matrix. Here we follow this idea by introducing two novel components into the current practice. First, a non-invasive method for online monitoring of resulting fluid dynamical characteristics of the coronary system is demonstrated for application during the perfusion decellularization of whole hearts. Second, the observation of the underlying rheological characteristics of the perfusates is employed to detect ongoing progress and maturation of the decellularization process. Measured data were contrasted to the respective release of specific cellular components. We demonstrate rheological measurements to be capable of detecting cellular debris along with a discriminative capture of DNA and protein ratios. We demonstrate that this perfusate biomass is well correlated to the biomass loss in the extracellular matrix produced by decellularization. The appearance of biomass components in the perfusates could specifically reflect the appearance of fluid dynamical characteristics that we monitored during the decellularization process. As rheological measuring of perfusate samples can be done within minutes, without any time-consuming preparation steps, we predict this to be a promising novel analytic strategy to control decellularization protocols, in time, by the actual conditions of the processed organ.

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.

Shear-related fibrillogenesis of fibronectin.

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.

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

Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbß3, αvß3, and α5ß1 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. ß3 integrins had a major impact on the interaction of FN on platelets. αvß3 showed a similar contribution to the binding of FN as αIIbß3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5ß1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than ß3 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 αIIbß3, αvß3, and α5ß1 or another still unknown receptor can be substituted.

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.

Highly efficient lentiviral transduction of phenotypically and genotypically characterized endothelial progenitor cells from adult peripheral blood.

Postnatal vasculogenesis has been implicated as an important mechanism for neovascularization via bone marrow-derived endothelial progenitor cells (EPCs) circulating in peripheral blood. In preparation of the utilization of EPCs in clinical protocols, we have generated blood-derived EPCs according to two established protocols by culturing either nonadherent mononuclear cells on fibronectin or adherent mononuclear cells on collagen. To explore the feasibility of these EPCs for their potential clinical use as target cells for genetic transduction to enhance their thromboresistance, newly designed retroviral and lentiviral gene ontology expression vectors were tested. Whereas cell clusters derived from the nonadherent cells demonstrated an only limited proliferative potential, cell colonies derived from collagen-adherent cells expanded more than a million-fold. Characterization of the exponentially growing cells by surface antigen and gene expression profiling revealed a consistently strong expression of characteristic endothelial markers, whereas expression of leukocyte markers was gradually lost. Using a single-step transduction protocol, we were able to achieve gene transfer efficiency of up to 99%. Our results suggest that the generated blood-derived EPC population might be attractive target cells for tissue engineering and gene therapy protocols due to their well defined phenotype, extensive proliferative potential, and efficient genetic transducibility, three important qualities that need to be defined prior to any clinical use.

Transforming growth factor-beta1 enhances the antifibrinolytic and prothrombotic state of growing endothelial cells in a cell cycle-specific manner.

Cell cycle-dependent modulation of protein expression may influence the balance of antithrombotic and prothrombotic properties of endothelial cells. In the present study, we examined the regulation of prothrombotic and antithrombotic molecules by transforming growth factor-beta1 (TGF-beta1) during distinct phases of the cell cycle in human umbilical vein endothelial cells. In the absence of TGF-beta1, the expression of thrombomodulin, the plasminogen activators u-PA and t-PA, and their inhibitor PAI-1 was significantly increased in the S/G2 compared to the G1 phase. Treatment of endothelial cells with TGF-beta1, however, resulted in elevated expression of PAI-1 specifically in the S/G2 phase, while t-PA and u-PA increased to the same extent in both the G1 and S/G2 phase. These findings demonstrate that the expression of a subset of hemostatically relevant proteins is regulated during endothelial cell cycle and that TGF-beta1 can differentially modulate cell cycle-controlled protein expression.

A Candida albicans chaperonin subunit (CaCct8p) as a suppressor of morphogenesis and Ras phenotypes in C. albicans and Saccharomyces cerevisiae.

Saccharomyces cerevisiae and the pathogen Candida albicans can be induced to undergo morphogenesis from a yeast to a filamentous form. A C. albicans gene (CaCCT8) was identified encoding a subunit of the Cct chaperonin complex, whose expression prevents filament formation in both fungi without interfering with growth of the yeast form. In S. cerevisiae, pseudohyphal growth induced by Ras2Val19, by overproduction of Phd1p or by expression of the C. albicans EFG1 gene, was blocked by CaCct8p and its N-terminally deleted derivative CaCct8-delta1p; in contrast, pseudohyphal induction by other components (Cph1p, Cdc42p) could not be suppressed, indicating that morphogenesis per se is not inhibited. CaCCT8 expression also interfered with other Ras2pVal19 phenotypes, including heat sensitivity, lack of glycogen accumulation and lack of sporulation. In C. albicans, overproduction of CaCct8p effectively blocked hyphal morphogenesis induced by starvation conditions and by serum. The results suggest that the activity of a component in the Ras2p signal transduction pathway is suppressed by excess chaperonin subunits. This component may be a novel folding target for the Cct complex. In agreement with this hypothesis, disruption of one of the two CaCCT8 alleles in C. albicans led to defective hyphal morphogenesis.