Dmitry Shvartsman

Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate

Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular matrix mechanics affect stem cell fate in 3D micro-environments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem cell (MSC) populations changes in response to the rigidity of 3D micro-environments, with osteogenesis occurring predominantly at 11–30 kPa. In contrast to previous 2D work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction-dependent and correlated with osteogenic commitment of MSC populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feedback to manipulate their fate.

Functional muscle regeneration with combined delivery of angiogenesis and myogenesis factors

Regenerative efforts typically focus on the delivery of single factors, but it is likely that multiple factors regulating distinct aspects of the regenerative process (e.g., vascularization and stem cell activation) can be used in parallel to affect regeneration of functional tissues. This possibility was addressed in the context of ischemic muscle injury, which typically leads to necrosis and loss of tissue and function. The role of sustained delivery, via injectable gel, of a combination of VEGF to promote angiogenesis and insulin-like growth factor-1 (IGF1) to directly promote muscle regeneration and the return of muscle function in ischemic rodent hindlimbs was investigated. Sustained VEGF delivery alone led to neoangiogenesis in ischemic limbs, with complete return of tissue perfusion to normal levels by 3 weeks, as well as protection from hypoxia and tissue necrosis, leading to an improvement in muscle contractility. Sustained IGF1 delivery alone was found to enhance muscle fiber regeneration and protected cells from apoptosis. However, the combined delivery of VEGF and IGF1 led to parallel angiogenesis, reinnervation, and myogenesis; as satellite cell activation and proliferation was stimulated, cells were protected from apoptosis, the inflammatory response was muted, and highly functional muscle tissue was formed. In contrast, bolus delivery of factors did not have any benefit in terms of neoangiogenesis and perfusion and had minimal effect on muscle regeneration. These results support the utility of simultaneously targeting distinct aspects of the regenerative process.

Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts

Lipid rafts play important roles in cellular functions through concentrating or sequestering membrane proteins. This requires proteins to differ in the stability of their interactions with lipid rafts. However, knowledge of the dynamics of membrane protein–raft interactions is lacking. We employed FRAP to measure in live cells the lateral diffusion of influenza hemagglutinin (HA) proteins that differ in raft association. This approach can detect weak interactions with rafts not detectable by biochemical methods. Wild-type (wt) HA and glycosylphosphatidylinositol (GPI)-anchored HA (BHA-PI) diffused slower than a nonraft HA mutant, but became equal to the latter after cholesterol depletion. When antigenically distinct BHA-PI and wt HA were coexpressed, aggregation of BHA-PI into immobile patches reduced wt HA diffusion rate, suggesting transient interactions with BHA-PI raft patches. Conversely, patching wt HA reduced the mobile fraction of BHA-PI, indicating stable interactions with wt HA patches. Thus, the anchoring mode determines protein–raft interaction dynamics. GPI-anchored and transmembrane proteins can share the same rafts, and different proteins can interact stably or transiently with the same raft domains.

Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies.

Fluorescence imaging of two independently labelled proteins is commonly used to determine their co‐localization in cells. Antibody‐mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co‐localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co‐localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. green‐ and red‐labelled) are co‐localized. Two methods are used to determine co‐localization. The ‘overlap’ analysis defines two objects as co‐localized if the centre of mass of one falls within the area of the other. The ‘nearest‐neighbour distance’ analysis considers two objects as co‐localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co‐patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP‐2 adaptor complexes. The approach presented is potentially applicable to studies of co‐localization by other methods (e.g. electron microscopy), and the nearest‐neighbour distance method can also be adapted to study phenomena of correlated placement.

Targeted Delivery of Nanoparticles to Ischemic Muscle for Imaging and Therapeutic Angiogenesis

Targeting of nanoparticles to ischemic tissues was studied in a murine ischemic hindlimb model. Intravenously injected fluorescent nanoparticles allowed ischemia-targeted imaging of ischemic muscles due to increased permeability of blood vessels in hypoxic tissues. Targeting efficiency correlated with blood perfusion after induction of ischemia and was enhanced in early stages of ischemia (<7 days). Therapeutic delivery of vascular endothelial growth factor (VEGF) was achieved by VEGF-conjugated nanoparticles and resulted in a 1.7-fold increase in blood perfusion, as compared to control mice. This work supports the application of nanoparticles as imaging and therapeutic modalities for ischemia treatment.

Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart

Cell delivery to the infarcted heart has emerged as a promising therapy, but is limited by very low acute retention and engraftment of cells. The objective of this study was to compare a panel of biomaterials to evaluate if acute retention can be improved with a biomaterial carrier. Cells were quantified post-implantation in a rat myocardial infarct model in five groups (n = 7-8); saline injection (current clinical standard), two injectable hydrogels (alginate, chitosan/β-glycerophosphate (chitosan/ß-GP)) and two epicardial patches (alginate, collagen). Human mesenchymal stem cells (hMSCs) were delivered to the infarct border zone with each biomaterial. At 24 h, retained cells were quantified by fluorescence. All biomaterials produced superior fluorescence to saline control, with approximately 8- and 14-fold increases with alginate and chitosan/β-GP injectables, and 47 and 59-fold increases achieved with collagen and alginate patches, respectively. Immunohistochemical analysis qualitatively confirmed these findings. All four biomaterials retained 50-60% of cells that were present immediately following transplantation, compared to 10% for the saline control. In conclusion, all four biomaterials were demonstrated to more efficiently deliver and retain cells when compared to a saline control. Biomaterial-based delivery approaches show promise for future development of efficient in vivo delivery techniques.

Clustering of Raft-Associated Proteins in the External Membrane Leaflet Modulates Internal Leaflet H-Ras Diffusion and Signaling

ABSTRACT One of the least-explored aspects of cholesterol-enriched domains (rafts) in cells is the coupling between such domains in the external and internal monolayers and its potential to modulate transbilayer signal transduction. Here, we employed fluorescence recovery after photobleaching to study the effects of antibody-mediated patching of influenza hemagglutinin (HA) proteins [raft-resident wild-type HA and glycosylphosphatidylinositol-anchored HA, or the nonraft mutant HA(2A520)] on the lateral diffusion of internal-leaflet raft and nonraft Ras isoforms (H-Ras and K-Ras, respectively). Our studies demonstrate that the clustering of outer-leaflet or transmembrane raft-associated HA proteins (but not their nonraft mutants) retards the lateral diffusion of H-Ras (but not K-Ras), suggesting stabilized interactions of H-Ras with the clusters of raft-associated HA proteins. These modulations were paralleled by specific effects on the activity of H-Ras but not of the nonraft K-Ras. Thus, clustering raft-associated HA proteins facilitated the early step whereby H-Ras is converted to an activated, GTP-loaded state but inhibited the ensuing step of downstream signaling via the Mek/Erk pathway. We propose a model for the modulation of transbilayer signaling by clustering of raft proteins, where external clustering (antibody or ligand mediated) enhances the association of internal-leaflet proteins with the stabilized clusters, promoting either enhancement or inhibition of signaling.

Cyclodextrins but not Compactin Inhibit the Lateral Diffusion of Membrane Proteins Independent of Cholesterol

Cholesterol and glycosphingolipid‐enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl‐β‐cyclodextrin (MβCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MβCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MβCD and by metabolic inhibition (compactin), and of treatment with α‐CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MβCD treatment retarded the lateral diffusion of both raft and non‐raft mutants, whereas similar cholesterol reduction (30–33%) by metabolic inhibition enhanced selectively the diffusion of the raft‐associated mutants. Moreover, α‐CD also inhibited the diffusion of raft and non‐raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol‐independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non‐raft proteins.

Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets

Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.

Sustained Delivery of VEGF Maintains Innervation and Promotes Reperfusion in Ischemic Skeletal Muscles Via NGF/GDNF Signaling

Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.