A scalable device-less biomaterial approach for subcutaneous islet transplantation.

The subcutaneous space has been shown to be a suitable site for islet transplantation, however an abundance of islets is required to achieve normoglycemia, often requiring multiple donors. The loss of islets is due to the hypoxic conditions islets experience during revascularization, resulting in apoptosis. Therefore, to reduce the therapeutic dosage required to achieve normoglycemia, pre-vascularization of the subcutaneous space has been pursued. In this study, we highlight a biomaterial-based approach using a methacrylic acid copolymer coating to generate a robust pre-vascularized subcutaneous cavity for islet transplantation. We also devised a simple, but not-trivial, procedure for filling the cavity with an islet suspension in collagen. We show that the pre-vascularized site can support a marginal mass of islets to rapidly return streptozotocin-induced diabetic SCID/bg mice to normoglycemia. Furthermore, immunocompetent Sprague Daley rats remained normoglycemia for up to 70 days until they experienced graft destabilization as they outgrew their implants. This work highlights methacrylic acid-based biomaterials as a suitable pre-vascularization strategy for the subcutaneous space that is scalable and doesn't require exogenous cells or growth factors.

The role of insulin growth factor-1 on the vascular regenerative effect of MAA coated disks and macrophage-endothelial cell crosstalk.

The IGF-1 signaling pathway and IGF-1-dependent macrophage/endothelial cell crosstalk was found to be critical features of the vascular regenerative effect displayed by implanted methacrylic acid -co-isodecyl acrylate (MAA-co-IDA; 40% MAA) coated disks in CD1 mice. Inhibition of IGF-1 signaling using AG1024 an IGF1-R tyrosine kinase inhibitor abrogated vessel formation 14 days after disk implantation in a subcutaneous pocket. Explanted tissue had increased arginase 1 expression and reduced iNOS expression consistent with the greater shift from "M1" ("pro-inflammatory") macrophages to "M2" ("pro-angiogenic") macrophages for MAA coated disks relative to control MM (methyl methacrylate-co-IDA) disks; the latter did not generate a vascular response and the polarization shift was muted with AG1024. In vitro, medium conditioned by macrophages (both human dTHP1 cells and mouse bone marrow derived macrophages) had elevated IGF-1 mRNA and protein levels, while the cells had reduced IGF1-R but elevated IGFBP-3 mRNA levels. These cells also had reduced iNOS and elevated Arg1 expression, consistent with the in vivo polarization results, including the inhibitory effects of AG1024. On the other hand, HUVEC exposed to dTHP1 conditioned medium migrated and proliferated faster suggesting that the primary target of the macrophage released IGF-1 was endothelial cells. Although further investigation is warranted, IGF-1 appears to be a key feature underpinning the observed vascularization. Why MAA based materials have this effect remains to be defined, however.

The α11 integrin mediates fibroblast-extracellular matrix-cardiomyocyte interactions in health and disease.

Excessive cardiac interstitial fibrosis impairs normal cardiac function. We have shown that the α11ß1 (α11) integrin mediates fibrotic responses to glycated collagen in rat myocardium by a pathway involving transforming growth factor-ß. Little is known of the role of the α11 integrin in the developing mammalian heart. Therefore, we examined the impact of deletion of the α11 integrin in wild-type mice and in mice treated with streptozotocin (STZ) to elucidate the role of the α11 integrin in normal cardiac homeostasis and in the pathogenesis of diabetes-related fibrosis. As anticipated, cardiac fibrosis was reduced in α11 integrin knockout mice (α11(-/-); C57BL/6 background) treated with STZ compared with STZ-treated wild-type mice (P < 0.05). Unexpectedly, diastolic function was impaired in both vehicle and STZ-treated α11(-/-) mice, as shown by the decreased minimum rate of pressure change and prolonged time constant of relaxation in association with increased end-diastolic pressure (all P < 0.05 compared with wild-type mice). Accordingly, we examined the phenotype of untreated α11(-/-) mice, which demonstrated a reduced cardiomyocyte cross-sectional cell area and myofibril thickness (all P < 0.05 compared with wild-type mice) and impaired myofibril arrangement. Immunostaining for desmin and connexin 43 showed abnormal intermediate filament organization at intercalated disks and impaired gap-junction development. Overall, deletion of the α11 integrin attenuates cardiac fibrosis in the mammalian mouse heart and reduces ECM formation as a result of diabetes. Furthermore, α11 integrin deletion impairs cardiac function and alters cardiomyocyte morphology. These findings shed further light on the poorly understood interaction between the fibroblast-cardiomyocyte and the ECM.

Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages.

An unbiased phosphoproteomic method was used to identify biomaterial-associated changes in the phosphorylation patterns of macrophage-like cells. The phosphorylation differences between differentiated THP1 (dTHP1) cells treated for 10, 20, or 30 min with a vascular regenerative methacrylic acid (MAA) copolymer or a control methyl methacrylate (MM) copolymer were determined by MS. There were 1,470 peptides (corresponding to 729 proteins) that were differentially phosphorylated in dTHP1 cells treated with the two materials with a greater cellular response to MAA treatment. In addition to identifying pathways (such as integrin signaling and cytoskeletal arrangement) that are well known to change with cell-material interaction, previously unidentified pathways, such as apoptosis and mRNA splicing, were also discovered.

Glycated Collagen Induces α11 Integrin Expression Through TGF-ß2 and Smad3.

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the α11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates α11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased α11 integrin and α-smooth muscle actin expression. Compared with native collagen, binding of purified α11ß1 integrin to glycated collagen was reduced by >fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-ß2 but not TGF-ß1 or TGF-ß3. The increased expression of TGF-ß2 was inhibited by triple helical collagen peptides that mimic the α11ß1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with α11 integrin luciferase promoter constructs, glycated collagen activated the α11 integrin promoter. Analysis of α11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between -809 and -1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-ß2 signaling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis.

α11 integrin stimulates myofibroblast differentiation in diabetic cardiomyopathy.

AIMS: Diabetic cardiomyopathy is characterized by the production of a disorganized fibrotic matrix in the absence of coronary atherosclerosis and hypertension. We examined whether adhesion of cardiac fibroblasts to glycated collagens mediates the differentiation of pro-fibrotic myofibroblasts, which may contribute to cardiac fibrosis. METHODS AND RESULTS: By microarray, we found that methylglyoxal-treated collagen selectively enhanced α11 integrin expression in human cardiac fibroblasts, while levels of other collagen-binding integrins (α1, α2, and α10) were unchanged. Similar increases in α11 integrin mRNA and protein expression were observed in cardiac fibroblasts from streptozotocin (STZ)-treated Sprague-Dawley rats. In human cardiac fibroblasts plated on methyglyoxal-treated collagen and in cardiac fibroblasts from diabetic rats, transforming growth factor (TGF)-ß2 but not TGF-ß1 or TGF-ß3 was increased compared with controls. Knock-down of α11 integrin and TGF-ß receptors with small-interfering RNA blocked the increased expression of TGF-ß2, α-smooth muscle actin (α-SMA), and α11 integrin that were induced in cells plated on methylglyoxal-treated collagen. Further, inhibition of Smad3 signalling blocked methylglyoxal-collagen up-regulation of α11 integrin and α-SMA expression. Rats with STZ-induced diabetes exhibited increased phosphorylation of Smad3 in cardiac tissues compared with control rats. CONCLUSION: Interactions between α11 integrins and the Smad-dependent TGF-ß2 signalling may contribute to the formation of pro-fibrotic myofibroblasts and the development of a fibrotic interstitium in diabetic cardiomyopathy.

Importance of protein-tyrosine phosphatase-alpha catalytic domains for interactions with SHP-2 and interleukin-1-induced matrix metalloproteinase-3 expression.

Interleukin-1 (IL-1) induces extracellular matrix degradation as a result of increased expression of matrix metalloproteinases (MMPs). We examined adhesion-restricted signaling pathways that enable IL-1-induced MMP release in human gingival and murine fibroblasts. Of the seven MMPs and three tissue inhibitors of MMPs screened, IL-1 enhanced release only of MMP3 when cells formed focal adhesions. Inhibition of protein-tyrosine phosphatases (PTPs), which are enriched in focal adhesions, blocked IL-1-induced MMP3 release. Accordingly, in contrast to wild-type cells, fibroblasts null for PTPalpha did not exhibit IL-1-induced MMP3 release. IL-1 treatment enhanced the recruitment of SHP-2 and PTPalpha to focal adhesions and the association of PTPalpha with SHP-2. Pulldown assays confirmed a direct interaction between PTPalpha and SHP-2, which was dependent on the intact, membrane-proximal phosphatase domain of PTPalpha. Interactions between SHP-2 and PTPalpha, recruitment of SHP-2 to focal adhesions, IL-1-induced ERK activation, and MMP3 expression were all blocked by point mutations in the phosphatase domains of PTPalpha. These data indicate that IL-1-induced signaling through focal adhesions leading to MMP3 release and interactions between SHP-2 and PTPalpha are dependent on the integrity of the catalytic domains of PTPalpha.

GAPDH binds GLUT4 reciprocally to hexokinase-II and regulates glucose transport activity.

Dietary glucose is taken up by skeletal muscle through GLUT4 (glucose transporter 4). We recently identified by MS proteins displaying insulin-dependent co-precipitation with Myc-tagged GLUT4 from L6 myotubes, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HKII (hexokinase-II). In the present paper we explored whether GAPDH and HKII interact directly with cytoplasmic regions of GLUT4 and their possible inter-relationship. Endogenous and recombinant GAPDH and HKII bound to a chimeric protein linearly encoding all three cytosolic domains of GLUT4 [GST (glutathione-transferase)-GLUT4-cyto]. Both proteins bound to a lesser extent the middle cytosolic loop but not individual N- or C-terminal domains of GLUT4. Purified GAPDH and HKII competed for binding to GST-GLUT4-cyto; ATP increased GAPDH binding and decreased HKII binding to this construct. The physiological significance of the GAPDH-GLUT4 interaction was explored by siRNA (small interfering RNA)-mediated GAPDH knockdown. Reducing GAPDH expression by 70% increased HKII co-precipitation with GLUT4-Myc from L6 cell lysates. GAPDH knockdown had no effect on surface-exposed GLUT4-Myc in basal or insulin-stimulated cells, but markedly and selectively diminished insulin-stimulated 3-O-methyl glucose uptake and GLUT4-Myc photolabelling with ATB-BMPA {2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)-2-propylamine}, suggesting that the exofacial glucose-binding site was inaccessible. The results show that GAPDH and HKII reciprocally interact with GLUT4 and suggest that these interactions regulate GLUT4 intrinsic activity in response to insulin.

GLUT4 vesicle recruitment and fusion are differentially regulated by Rac, AS160, and Rab8A in muscle cells.

Insulin increases glucose uptake into muscle by enhancing the surface recycling of GLUT4 transporters. In myoblasts, insulin signals bifurcate downstream of phosphatidylinositol 3-kinase into separate Akt and Rac/actin arms. Akt-mediated Rab-GAP AS160 phosphorylation and Rac/actin are required for net insulin gain of GLUT4, but the specific steps (vesicle recruitment, docking or fusion) regulated by Rac, actin dynamics, and AS160 target Rab8A are unknown. In L6 myoblasts expressing GLUT4myc, blocking vesicle fusion by tetanus toxin cleavage of VAMP2 impeded GLUT4myc membrane insertion without diminishing its build-up at the cell periphery. Conversely, actin disruption by dominant negative Rac or Latrunculin B abolished insulin-induced surface and submembrane GLUT4myc accumulation. Expression of non-phosphorylatable AS160 (AS160-4P) abrogated membrane insertion of GLUT4myc and partially reduced its cortical build-up, an effect magnified by selective Rab8A knockdown. We propose that insulin-induced actin dynamics participates in GLUT4myc vesicle retention beneath the membrane, whereas AS160 phosphorylation is essential for GLUT4myc vesicle-membrane docking/fusion and also contributes to GLUT4myc cortical availability through Rab8A.

Alpha-actinin-4 is selectively required for insulin-induced GLUT4 translocation.

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64-75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of alpha-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton.