Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist.

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against αv and ß3 but not α5 or ß1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human αvß3 and αvß6 integrins, but not α5ß1 integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an αv-selective integrin agonist. This study provides evidence that cell surface αv-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

Assembly of fibronectin fibrils selectively attenuates platelet-derived growth factor-induced intracellular calcium release in fibroblasts.

Cellular responses to platelet-derived growth factor (PDGF) are altered in a variety of pathological conditions, including cancers, fibroses, and vascular diseases, making PDGF-induced signaling pathways important therapeutic targets. The limited success of therapies designed to impact PDGF pathways may be overcome with a clearer understanding of how cells integrate signals from PDGF and the extracellular matrix (ECM). Here, we assessed the effects of fibronectin matrix assembly on the responsiveness of mesenchymal cells to PDGF. Our results indicate that fibroblast-mediated assembly of fibronectin fibrils attenuates intracellular calcium release in response to PDGF. The dose-dependent inhibition of PDGF-induced intracellular calcium release was specific to the ECM form of fibronectin. Further, a recombinant protein engineered to mimic ECM fibronectin similarly attenuated intracellular calcium release in response to PDGF. Of note, fibronectin attenuated the PDGF-calcium signaling axis at the level of phosphoinositide 3-kinase (PI3K) activation. Interestingly, ECM fibronectin did not alter other intracellular signals activated by PDGF, including activation of PDGF receptor ß, AKT Ser/Thr kinase, phospholipase Cγ1, and extracellular signal-regulated kinase 1/2 (ERK1/2). Rather, fibronectin inhibited activation of the p55 regulatory subunit of PI3K in response to a variety of stimuli, indicating that ECM fibronectin selectively attenuates the intracellular calcium release cascade while leaving intact other PDGF signaling pathways. Selective regulation of calcium signaling by ECM fibronectin via the p55 regulatory subunit of PI3K represents a mechanism by which cells tune their response to PDGF and may therefore serve as a target to selectively regulate one branch of PDGF signaling.

Ultrasound patterning technologies for studying vascular morphogenesis in 3D.

Investigations in this report demonstrate the versatility of ultrasound-based patterning and imaging technologies for studying determinants of vascular morphogenesis in 3D environments. Forces associated with ultrasound standing wave fields (USWFs) were employed to non-invasively and volumetrically pattern endothelial cells within 3D collagen hydrogels. Patterned hydrogels were composed of parallel bands of endothelial cells located at nodal regions of the USWF and spaced at intervals equal to one half wavelength of the incident sound field. Acoustic parameters were adjusted to vary the spatial dimensions of the endothelial bands, and effects on microvessel morphogenesis were analyzed. High-frequency ultrasound imaging techniques were used to image and quantify the spacing, width and density of initial planar cell bands. Analysis of resultant microvessel networks showed that vessel width, orientation, density and branching activity were strongly influenced by the initial 3D organization of planar bands and, hence, could be controlled by acoustic parameters used for patterning. In summary, integration of USWF-patterning and high-frequency ultrasound imaging tools enabled fabrication of vascular constructs with defined microvessel size and orientation, providing insight into how spatial cues in 3D influence vascular morphogenesis.

Extracellular matrix fibronectin initiates endothelium-dependent arteriolar dilatation via the heparin-binding, matricryptic RWRPK sequence of the first type III repeat of fibrillar fibronectin.

KEY POINTS: The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms. In addition to the many metabolic signals that mediate this vasodilatation, we show here that the extracellular matrix protein fibronectin also contributes to the response. This vasodilatory signal requires the heparin-binding matricryptic RWRPK sequence in the first type III repeat of fibrillar fibronectin. The fibronectin-dependent component of the integrated muscle contraction-dependent arteriolar vasodilatation is coupled through an endothelial cell-dependent signalling pathway. Recent studies in contracting skeletal muscle have shown that functional vasodilatation in resistance arterioles has an endothelial cell (EC)-dependent component, and, separately have shown that the extracellular matrix protein fibronectin (FN) contributes to functional dilatation in these arterioles. Here we test the hypotheses that (i) the matricryptic heparin-binding region of the first type III repeat of fibrillar FN (FNIII1H) mediates vasodilatation, and (ii) this response is EC dependent. Engineered FN fragments with differing (defined) heparin- and integrin-binding capacities were applied directly to resistance arterioles in cremaster muscles of anaesthetized (pentobarbital sodium, 65 mg kg(-1)) mice. Both FNIII1H,8-10 and FNIII1H induced dilatations (12.2 ± 1.7 µm, n = 12 and 17.2 ± 2.4 µm, n = 14, respectively) whereas mutation of the active sequence (R(613) WRPK) of the heparin binding region significantly diminished the dilatation (3.2 ± 1.8 µm, n = 10). Contraction of skeletal muscle fibres via electrical field stimulation produced a vasodilatation (19.4 ± 1.2 µm, n = 12) that was significantly decreased (to 7.0 ± 2.7 µm, n = 7, P < 0.05) in the presence of FNIII1Peptide 6, which blocks extracellular matrix (ECM) FN and FNIII1H signalling. Furthermore, FNIII1H,8-10 and FNIII1H applied to EC-denuded arterioles failed to produce any dilatation indicating that endothelium was required for the response. Finally, FNIII1H significantly increased EC Ca(2+) (relative fluorescence 0.98 ± 0.02 in controls versus 1.12 ± 0.05, n = 17, P < 0.05). Thus, we conclude that ECM FN-dependent vasodilatation is mediated by the heparin-binding (RWRPK) sequence of FNIII1 in an EC-dependent manner. Importantly, blocking this signalling sequence decreased the dilatation to skeletal muscle contraction, indicating that there is a physiological role for this FN-dependent mechanism.

Extracellular matrix fibronectin mediates an endothelial cell response to shear stress via the heparin-binding, matricryptic RWRPK sequence of FNIII1H.

Endothelial cells (EC) respond to mechanical forces such as shear stress in a variety of ways, one of which is cytoskeletal realignment in the direction of flow. Our earlier studies implicated the extracellular matrix protein fibronectin in mechanosensory signaling to ECs in intact arterioles, via a signaling pathway dependent on the heparin-binding region of the first type III repeat of fibrillar fibronectin (FNIII1H). Here we test the hypothesis that FNIII1H is required for EC stress fiber realignment under flow. Human umbilical vein ECs (HUVECs) exposed to defined flow conditions were used as a well-characterized model of this stress fiber alignment response. Our results directly implicate FNIII1H in realignment of stress fibers in HUVECs and, importantly, show that the matricryptic heparin-binding RWRPK sequence located in FNIII1 is required for the response. Furthermore, we show that flow-mediated stress fiber realignment in ECs adhered via α5ß1-integrin-specific ligands does not occur in the absence of FHIII1H, whereas, in contrast, αvß3-integrin-mediated stress fiber realignment under flow does not require FNIII1H. Our findings thus indicate that there are two separate mechanosignaling pathways mediating the alignment of stress fibers after exposure of ECs to flow, one dependent on αvß3-integrins and one dependent on FNIII1H. This study strongly supports the conclusion that the RWRPK region of FNIII1H may have broad capability as a mechanosensory signaling site.

Scholte wave generation during single tracking location shear wave elasticity imaging of engineered tissues.

The physical environment of engineered tissues can influence cellular functions that are important for tissue regeneration. Thus, there is a critical need for noninvasive technologies capable of monitoring mechanical properties of engineered tissues during fabrication and development. This work investigates the feasibility of using single tracking location shear wave elasticity imaging (STL-SWEI) for quantifying the shear moduli of tissue-mimicking phantoms and engineered tissues in tissue engineering environments. Scholte surface waves were observed when STL-SWEI was performed through a fluid standoff, and confounded shear moduli estimates leading to an underestimation of moduli in regions near the fluid-tissue interface.

Spatial patterning of endothelial cells and vascular network formation using ultrasound standing wave fields.

The spatial organization of cells is essential for proper tissue assembly and organ function. Thus, successful engineering of complex tissues and organs requires methods to control cell organization in three dimensions. In particular, technologies that facilitate endothelial cell alignment and vascular network formation in three-dimensional tissue constructs would provide a means to supply essential oxygen and nutrients to newly forming tissue. Acoustic radiation forces associated with ultrasound standing wave fields can rapidly and non-invasively organize cells into distinct multicellular planar bands within three-dimensional collagen gels. Results presented herein demonstrate that the spatial pattern of endothelial cells within three-dimensional collagen gels can be controlled by design of acoustic parameters of the sound field. Different ultrasound standing wave field exposure parameters were used to organize endothelial cells into either loosely aggregated or densely packed planar bands. The rate of vessel formation and the morphology of the resulting endothelial cell networks were affected by the initial density of the ultrasound-induced planar bands of cells. Ultrasound standing wave fields provide a rapid, non-invasive approach to pattern cells in three-dimensions and direct vascular network formation and morphology within engineered tissue constructs.

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound.

Type I collagen is the primary fibrillar component of the extracellular matrix, and functional properties of collagen arise from variations in fiber structure. This study investigated the ability of ultrasound to control collagen microstructure during hydrogel fabrication. Under appropriate conditions, ultrasound exposure of type I collagen during polymerization altered fiber microstructure. Scanning electron microscopy and second-harmonic generation microscopy revealed decreased collagen fiber diameters in response to ultrasound compared to sham-exposed samples. Results of mechanistic investigations were consistent with a thermal mechanism for the effects of ultrasound on collagen fiber structure. To control collagen microstructure site-specifically, a high frequency, 8.3-MHz, ultrasound beam was directed within the center of a large collagen sample producing dense networks of short, thin collagen fibrils within the central core of the gel and longer, thicker fibers outside the beam area. Fibroblasts seeded onto these gels migrated rapidly into small, circularly arranged aggregates only within the beam area, and clustered fibroblasts remodeled the central, ultrasound-exposed collagen fibrils into dense sheets. These investigations demonstrate the capability of ultrasound to spatially pattern various collagen microstructures within an engineered tissue noninvasively, thus enhancing the level of complexity of extracellular matrix microenvironments and cellular functions achievable within three-dimensional engineered tissues.

In vivo acoustic patterning of endothelial cells for tissue vascularization.

Strategies to fabricate microvascular networks that structurally and functionally mimic native microvessels are needed to address a host of clinical conditions associated with tissue ischemia. The objective of this work was to advance a novel ultrasound technology to fabricate complex, functional microvascular networks directly in vivo. Acoustic patterning utilizes forces within an ultrasound standing wave field (USWF) to organize cells or microparticles volumetrically into defined geometric assemblies. A dual-transducer system was developed to generate USWFs site-specifically in vivo through interference of two ultrasound fields. The system rapidly patterned injected cells or microparticles into parallel sheets within collagen hydrogels in vivo. Acoustic patterning of injected endothelial cells within flanks of immunodeficient mice gave rise to perfused microvessels within 7 days of patterning, whereas non-patterned cells did not survive. Thus, externally-applied ultrasound fields guided injected endothelial cells to self-assemble into perfused microvascular networks in vivo. These studies advance acoustic patterning towards in vivo tissue engineering by providing the first proof-of-concept demonstration that non-invasive, ultrasound-mediated cell patterning can be used to fabricate functional microvascular networks directly in vivo.

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly.

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair. Cross-talk between cell-cell and cell-extracellular matrix adhesion complexes is essential for the organization of cells into complex, functional tissue during embryonic development and tissue remodeling. Here, we demonstrate that fibronectin matrix assembly affects the organization, composition, and function of N-cadherin-based adherens junctions. Using fibronectin-null mouse embryonic myofibroblasts, we identified a novel quaternary complex composed of N-cadherin, ß-catenin, tensin, and actin that exists in the absence of a fibronectin matrix. In the absence of fibronectin, homophilic N-cadherin ligation recruited both tensin and α5ß1 integrins into nascent cell-cell adhesions. Initiation of fibronectin matrix assembly disrupted the association of tensin and actin with N-cadherin, released α5ß1 integrins and tensin from cell-cell contacts, stimulated N-cadherin reorganization into thin cellular protrusions, and decreased N-cadherin adhesion. Fibronectin matrix assembly has been shown to recruit α5ß1 integrins and tensin into fibrillar adhesions. Taken together, these studies suggest that tensin serves as a common cytoskeletal link for integrin- and cadherin-based adhesions and that the translocation of α5ß1 integrins from cell-cell contacts into fibrillar adhesions during fibronectin matrix assembly is a novel mechanism by which cell-cell and cell-matrix adhesions are coordinated.

Receptor binding domain of SARS-CoV-2 is a functional αv-integrin agonist.

Among the novel mutations distinguishing SARS-CoV-2 from similar respiratory coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2 (ACE2)-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. In the present study, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared to RGD-containing, integrin-binding fragments of fibronectin. S1-RBD supported adhesion of both fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells. Cell adhesion to S1-RBD was cation- and RGD-dependent, and was inhibited by blocking antibodies against α v and ß 3 , but not α 5 or ß 1 , integrins. Similarly, direct binding of S1-RBD to recombinant human α v ß 3 and α v ß 6 integrins, but not α 5 ß 1 integrins, was observed by surface plasmon resonance. Adhesion to S1-RBD initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin, Akt activation, and supported cell proliferation. Together, these data demonstrate that the RGD sequence within S1-RBD can function as an α v -selective integrin agonist. This study provides evidence that cell surface α v -containing integrins can respond functionally to spike protein and raise the possibility that S1-mediated dysregulation of ECM dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

Time- and Dose-Dependent Effects of Pulsed Ultrasound on Dermal Repair in Diabetic Mice.

Chronic wounds, including diabetic, leg and pressure ulcers, impose a significant health care burden worldwide. Some evidence indicates that ultrasound can enhance soft tissue repair. However, therapeutic responses vary among individuals, thereby limiting clinical translation. Here, effects of pulsed ultrasound on dermal wound healing were assessed using a murine model of chronic, diabetic wounds. An ultrasound exposure system was developed to provide daily ultrasound exposures to full-thickness, excisional wounds in genetically diabetic mice. Wounds were exposed to 1 MHz ultrasound (2 ms pulse, 100 Hz pulse repetition frequency, 0-0.4 MPa) for 2 or 3 wk. Granulation tissue thickness and wound re-epithelialization increased as a function of increasing ultrasound pressure amplitude. At 2 wk after injury, significant increases in granulation tissue thickness and epithelial ingrowth were observed in response to 1 MHz pulsed ultrasound at 0.4 MPa. Wounds exposed to 0.4 MPa ultrasound for 3 wk were characterized by collagen-dense, revascularized granulation tissue with a fully restored, mature epithelium. Of note, only half of wounds exposed to 0.4 MPa ultrasound showed significant granulation tissue deposition after 2 wk of treatment. Thus, the db+/db+ mouse model may help to identify biological variables that influence individual responses to pulsed ultrasound and accelerate clinical translation.

Extracellular matrix fibronectin mechanically couples skeletal muscle contraction with local vasodilation.

During exercise, local mechanisms in tissues cause arterioles to rapidly dilate to increase blood flow to tissues to meet the metabolic demands of contracting muscle. Despite decades of study, the mechanisms underlying this important aspect of blood flow control are still far from clear. We now report a novel mechanism wherein fibronectin fibrils in connective tissue matrices transduce signals from contracting skeletal muscle to local blood vessels to increase blood flow. Using intravital microscopy, we show that local vasodilation in response to skeletal muscle contraction is specifically inhibited by an antibody that recognizes the matricryptic site in the first type III repeat of fibronectin (FNIII-1). In the absence of skeletal muscle contraction, direct application of FNIII-1-containing fibronectin fragments to cremaster muscle arterioles in situ, triggered a rapid, specific, and reversible local dilation that was mediated by nitric oxide and required the cryptic, heparin-binding sequence of FNIII-1. Furthermore, application of function-blocking FNIII-1 peptides to cremaster muscle arterioles rapidly and specifically decreased their diameter, indicating that the matricryptic site of fibronectin also contributes to resting vascular tone. Alexa fluor 488-labeled fibronectin, administered intravenously, was rapidly assembled into elongated, branching fibrils in the extracellular matrix of intact cremaster muscle, demonstrating active polymerization of fibronectin in areas adjacent to blood vessels. Together, these data provide the first evidence that a matricryptic, heparin-binding site within fibronectin fibrils of adult connective tissue plays a dynamic role in regulating both vascular responses and vascular tone.

Acoustic Fabrication of Collagen-Fibronectin Composite Gels Accelerates Microtissue Formation.

Ultrasound can influence biological systems through several distinct acoustic mechanisms that can be manipulated by varying reaction conditions and acoustic exposure parameters. We recently reported a new ultrasound-based fabrication technology that exploits the ability of ultrasound to generate localized mechanical forces and thermal effects to control collagen fiber microstructure non-invasively. Exposing solutions of type I collagen to ultrasound during the period of microfibril assembly produced changes in collagen fiber structure and alignment, and increased the biological activity of the resultant collagen hydrogels. In the extracellular matrix, interactions between fibronectin and collagen fibrils influence the biological activity of both proteins. Thus, in the present study, we examined how addition of fibronectin to collagen solutions prior to ultrasound exposure affects protein organization and the biological activity of the composite hydrogels. Results indicate that ultrasound can alter the distribution of fibronectin within 3D hydrogels via thermal and non-thermal mechanisms to produce composite hydrogels that support accelerated microtissue formation. The use of acoustic energy to drive changes in protein conformation to functionalize biomaterials has much potential as a unique, non-invasive technology for tissue engineering and regenerative medicine.

Using Acoustic Fields to Fabricate ECM-Based Biomaterials for Regenerative Medicine Applications.

Ultrasound is emerging as a promising tool for both characterizing and fabricating engineered biomaterials. Ultrasound-based technologies offer a diverse toolbox with outstanding capacity for optimization and customization within a variety of therapeutic contexts, including improved extracellular matrix-based materials for regenerative medicine applications. Non-invasive ultrasound fabrication tools include the use of thermal and mechanical effects of acoustic waves to modify the structure and function of extracellular matrix scaffolds both directly, and indirectly via biochemical and cellular mediators. Materials derived from components of native extracellular matrix are an essential component of engineered biomaterials designed to stimulate cell and tissue functions and repair or replace injured tissues. Thus, continued investigations into biological and acoustic mechanisms by which ultrasound can be used to manipulate extracellular matrix components within three-dimensional hydrogels hold much potential to enable the production of improved biomaterials for clinical and research applications.

CD4+ T Cell Interstitial Migration Controlled by Fibronectin in the Inflamed Skin.

The extracellular matrix (ECM) is extensively remodeled during inflammation providing essential guidance cues for immune cell migration and signals for cell activation and survival. There is increasing interest in the therapeutic targeting of ECM to mitigate chronic inflammatory diseases and enhance access to the tumor microenvironment. T cells utilize the ECM as a scaffold for interstitial migration, dependent on T cell expression of matrix-binding integrins αVß1/αVß3 and tissue display of the respective RGD-containing ligands. The specific ECM components that control T cell migration are unclear. Fibronectin (FN), a canonical RGD-containing matrix component, is heavily upregulated in inflamed tissues and in vitro can serve as a substrate for leukocyte migration. However, limited by lack of tools to intravitally visualize and manipulate FN, the specific role of FN in effector T cell migration in vivo is unknown. Here, we utilize fluorescently-tagged FN to probe for FN deposition, and intravital multiphoton microscopy to visualize T cell migration relative to FN in the inflamed ear dermis. Th1 cells were found to migrate along FN fibers, with T cells appearing to actively push or pull against flexible FN fibers. To determine the importance of T cell interactions with FN, we used a specific inhibitor of FN polymerization, pUR4. Intradermal delivery of pUR4 (but not the control peptide) to the inflamed skin resulted in a local reduction in FN deposition. We also saw a striking attenuation of Th1 effector T cell movement at the pUR4 injection site, suggesting FN plays a key role in T cell interstitial migration. In mechanistic studies, pUR4 incubation with FN in vitro resulted in enhanced tethering of T cells to FN matrix, limiting productive migration. In vivo, such tethering led to increased Th1 accumulation in the inflamed dermis. Enhanced Th1 accumulation exacerbated inflammation with increased Th1 activation and IFNγ cytokine production. Thus, our studies highlight the importance of ECM FN fibrils for T cell migration in inflamed tissues and suggest that manipulating local levels of ECM FN may prove beneficial in promoting T cell accumulation in tissues and enhancing local immunity to infection or cancer.

A cryptic fragment from fibronectin's III1 module localizes to lipid rafts and stimulates cell growth and contractility.

The interaction of cells with the extracellular matrix (ECM) form of fibronectin (FN) triggers changes in growth, migration, and cytoskeletal organization that differ from those generated by soluble FN. As cells deposit and remodel their FN matrix, the exposure of new epitopes may serve to initiate responses unique to matrix FN. To determine whether a matricryptic site within the III1 module of FN modulates cell growth or cytoskeletal organization, a recombinant FN with properties of matrix FN was constructed by directly linking the cryptic, heparin-binding COOH-terminal fragment of III1 (III1H) to the integrin-binding III8-10 modules (glutathione-S-transferase [GST]-III1H,8-10). GST-III1H,8-10 specifically stimulated increases in cell growth and contractility; integrin ligation alone was ineffective. A construct lacking the integrin-binding domain (GST-III1H,2-4) retained the ability to stimulate cell contraction, but was unable to stimulate cell growth. Both GST-III1H,2-4 and matrix FN colocalized with caveolin and fractionated with low-density membrane complexes by a mechanism that required heparan sulfate proteoglycans. Disruption of caveolae inhibited the FN- and III1H-mediated increases in cell contraction and growth. These data suggest that a portion of ECM FN partitions into lipid rafts and differentially regulates cytoskeletal organization and growth, in part, through the exposure of a neoepitope within the conformationally labile III1 module.

A Matricryptic Conformation of the Integrin-Binding Domain of Fibronectin Regulates Platelet-Derived Growth Factor-Induced Intracellular Calcium Release.

Platelet-derived growth factor (PDGF) signaling is dysregulated in a wide variety of diseases, making PDGF an attractive therapeutic target. However, PDGF also affects numerous signaling cascades essential for tissue homeostasis, limiting the development of PDGF-based therapies that lack adverse side-effects. Recent studies showed that fibroblast-mediated assembly of extracellular matrix (ECM) fibronectin fibrils attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact. In the present study, a series of recombinant fibronectin-derived fusion proteins were used to localize the sequences in fibronectin that are responsible for this inhibition. Results demonstrate that attenuation of PDGF-induced intracellular calcium release by the fibronectin matrix mimetic, FNIII1H,8-10 requires α5ß1 integrin ligation, but is not dependent upon the matricryptic, heparin-binding site of FNIII1. Intact cell-binding fibronectin fragments were also unable to attenuate PDGF-induced intracellular calcium release. In contrast, a novel integrin-binding fragment that adopts an extended and aligned conformational state, inhibited both PI3K activation and intracellular calcium release in response to PDGF. Taken together, these studies provide evidence that attenuation of PDGF-induced intracellular calcium release by fibronectin is mediated by a novel conformation of the α5ß1 integrin-binding, FNIII9-10 modules, that is expressed by fibrillar fibronectin.

Non-invasive acoustic fabrication methods to enhance collagen hydrogel bioactivity.

Much attention has focused recently on utilizing components of the extracellular matrix (ECM) as natural building blocks for a variety of tissue engineering applications and regenerative medicine therapies. Consequently, new fabrication methods are being sought to enable molecular control over the structural characteristics of ECM molecules in order to improve their biological function. Exposing soluble collagen to acoustic forces associated with ultrasound propagation produces localized variations in collagen microfiber organization that in turn, promote cell behaviors essential for tissue regeneration, including cell migration and matrix remodeling. In the present study, mechanisms by which ultrasound interacts with polymerizing collagen to produce functional changes in collagen microstructure were investigated. The rate of collagen polymerization was manipulated by adjusting the pH of collagen solutions and the temperature at which gels were polymerized. Results demonstrate that the phase transition of type I collagen from fluid to gel triggered a simultaneous increase in acoustic absorption. This phase transition of collagen involves the lateral growth of early-stage collagen microfibrils and importantly, corresponded to a defined period of time during which exposure to ultrasound introduced both structural and functional changes to the resultant collagen hydrogels. Together, these experiments isolated a critical window in the collagen fiber assembly process during which mechanical forces associated with ultrasound propagation are effective in producing structural changes that underlie the ability of acoustically-modified collagen hydrogels to stimulate cell migration. These results demonstrate that changes in material properties associated with collagen polymerization are a fundamental component of the mechanism by which acoustic forces modify collagen biomaterials to enhance biological function.

Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions.

Remodeling of extracellular matrices occurs during development, wound healing, and in a variety of pathological processes including atherosclerosis, ischemic injury, and angiogenesis. Thus, identifying factors that control the balance between matrix deposition and degradation during tissue remodeling is essential for understanding mechanisms that regulate a variety of normal and pathological processes. Using fibronectin-null cells, we found that fibronectin polymerization into the extracellular matrix is required for the deposition of collagen-I and thrombospondin-1 and that the maintenance of extracellular matrix fibronectin fibrils requires the continual polymerization of a fibronectin matrix. Further, integrin ligation alone is not sufficient to maintain extracellular matrix fibronectin in the absence of fibronectin deposition. Our data also demonstrate that the retention of thrombospondin-1 and collagen I into fibrillar structures within the extracellular matrix depends on an intact fibronectin matrix. An intact fibronectin matrix is also critical for maintaining the composition of cell-matrix adhesion sites; in the absence of fibronectin and fibronectin polymerization, neither alpha5beta1 integrin nor tensin localize to fibrillar cell-matrix adhesion sites. These data indicate that fibronectin polymerization is a critical regulator of extracellular matrix organization and stability. The ability of fibronectin polymerization to act as a switch that controls the organization and composition of the extracellular matrix and cell-matrix adhesion sites provides cells with a means of precisely controlling cell-extracellular matrix signaling events that regulate many aspects of cell behavior including cell proliferation, migration, and differentiation.