Impact of integrin–matrix interaction and signaling on insulin gene expression and the mesenchymal transition of human β‐cells

A critical shortage of donor pancreata currently prevents the development of a universal cell‐based therapy for type I diabetes. The ex vivo expansion of insulin‐producing β‐cells offers a potential solution but is problematic due to the inherent tendency of these cells to transition into mesenchymal‐like cells that are devoid of function. Here, we demonstrate for the first time that exposure to elements of the extracellular matrix (ECM) directly potentiates the mesenchymal transition of cultured fetal β‐cells and causes associated declines in insulin gene expression. Individual ECM constituents varied in their ability to induce such responses, with collagen‐IV (C‐IV) and fibronectin inducing strong responses, whereas laminin‐1 had no significant effect. Mesenchymal transition and concomitant losses in insulin gene expression observed on C‐IV were found to be dependent on β₁‐integrin ligation and were augmented in the presence of hepatocyte growth factor. Importantly, selective inhibition of c‐Src, c‐Jun N‐terminal kinase (JNK), and extracellular signal‐regulated kinase (ERK) prior to exposure to C‐IV prevented mesenchymal transition and effectively preserved insulin expression. Fetal β‐cells undergoing mesenchymal transition were found to acquire α₁β₁ expression, and ligation of this integrin then promotes declines in insulin gene expression and a marked increase in β‐cell motility. Inhibition of Src‐, ERK‐, or JNK‐dependent signaling combined with the selective regulation of matrix exposure may ultimately facilitate the development of more effective β‐cell expansion protocols. J. Cell. Physiol. 224:101–111, 2010 © 2010 Wiley‐Liss, Inc.     

A new paradigm in cell therapy for diabetes: Turning pancreatic α‐cells into β‐cells

Cell therapy means treating diseases with the body's own cells. One of the cell types most in demand for therapeutic purposes is the pancreatic β‐cell. This is because diabetes is one of the major healthcare problems in the world. Diabetes can be treated by islet transplantation but the major limitation is the shortage of organ donors. To overcome the shortfall in donors, alternative sources of pancreatic β‐cells must be found. Potential sources include embryonic or adult stem cells or, from existing β‐cells. There is now a startling new addition to this list of therapies: the pancreatic α‐cell. Thorel and colleagues recently showed that under circumstances of extreme pancreatic β‐cell loss, α‐cells may serve to replenish the insulin‐producing compartment. This conversion of α‐cells to β‐cells represents an example of transdifferentiation. Understanding the molecular basis for transdifferentiation may help to enhance the generation of β‐cells for the treatment of diabetes.     

Obesity‐Induced Diabetes in Mouse Strains Treated With Gold Thioglucose: A Novel Animal Model for Studying β‐Cell Dysfunction

An obesity‐induced diabetes model using genetically normal mouse strains would be invaluable but remains to be established. One reason is that several normal mouse strains are resistant to high‐fat diet‐induced obesity. In the present study, we show the effectiveness of gold thioglucose (GTG) in inducing hyperphagia and severe obesity in mice, and demonstrate the development of obesity‐induced diabetes in genetically normal mouse strains. GTG treated DBA/2, C57BLKs, and BDF1 mice gained weight rapidly and exhibited significant increases in nonfasting plasma glucose levels 8–12 weeks after GTG treatment. These mice showed significantly impaired insulin secretion, particularly in the early phase after glucose load, and reduced insulin content in pancreatic islets. Interestingly, GTG treated C57BL/6 mice did not become diabetic and retained normal early insulin secretion and islet insulin content despite being as severely obese and insulin resistant as the other mice. These results suggest that the pathogenesis of obesity‐induced diabetes in GTG‐treated mice is attributable to the inability of their pancreatic β‐cells to secrete enough insulin to compensate for insulin resistance. Mice developing obesity‐induced diabetes after GTG treatment might be a valuable tool for investigating obesity‐induced diabetes. Furthermore, comparing the genetic backgrounds of mice with different susceptibilities to diabetes may lead to the identification of novel genetic factors influencing the ability of pancreatic β‐cells to secrete insulin.     

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

The interaction between uPAR and vitronectin triggers ligand‐independent adhesion signalling by integrins

The urokinase‐type plasminogen activator receptor (uPAR) is a non‐integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure–function analyses of uPAR, VN and integrins, we document that uPAR‐mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin–matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non‐canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non‐integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.     

Dual factor delivery of CXCL12 and Exendin‐4 for improved survival and function of encapsulated beta cells under hypoxic conditions

A bioartifical pancreas (BAP) remains a promising approach for treating insulin‐dependent diabetes. Several obstacles to the clinical implementation of a BAP remain, including hypoxia following implantation. Within native pancreatic islets, CXCL12 and glucagon‐like peptide‐1 (GLP‐1) act in a paracrine fashion to promote the survival, function, and proliferation of β‐cells. This work sought to investigate if the presentation of CXCL12 and delivery of a GLP‐1 receptor analog, Exendin‐4 (Ex‐4), alone and in combination, conferred pro‐survival and insulinotropic effects on an encapsulated β‐cell line, βTC‐tet, cultured under hypoxic conditions of 7.6 mmHg O₂. Our findings indicate that presentation of CXCL12 in the encapsulation matrix completely abrogated apoptosis under hypoxic conditions. Delivery of Ex‐4 increased insulin secretion rate under both normoxic and hypoxic conditions, and additionally reduced apoptosis under hypoxic conditions. Furthermore, presentation of CXCL12 combined with Ex‐4 delivery significantly increased insulin secretion rate under hypoxic conditions compared to delivery of Ex‐4 alone. These findings demonstrate that the presentation of CXCL12 combined with the delivery of Ex‐4 may constitute a promising strategy for supporting β‐cell function and survival following transplantation. Biotechnol. Bioeng. 2013; 110: 2292–2300. © 2013 Wiley Periodicals, Inc.     

Lentinan protects pancreatic β cells from STZ‐induced damage

Pancreatic β‐cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus (DM). In this study, we evaluated the effect of lentinan (LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β‐cell apoptosis and dysfunction caused by streptozotocin (STZ) and the possible mechanisms implicated. The rat insulinoma cell line INS‐1 were pre‐treated with the indicated concentration of LNT for 30 min. and then incubated for 24 hrs with or without 0.5 mM STZ. We found that STZ treatment causes apoptosis of INS‐1 cells by enhancement of intracellular reactive oxygen species (ROS) accumulation, inducible nitric oxide synthase (iNOS) expression and nitric oxide release and activation of the c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) signalling pathways. However, LNT significantly increased cell viability and effectively attenuated STZ‐induced ROS production, iNOS expression and nitric oxide release and the activation of JNK and p38 MAPK in a dose‐dependent manner in vitro. Moreover, LNT dose‐dependently prevented STZ‐induced inhibition of insulin synthesis by blocking the activation of nuclear factor kappa beta and increasing the level of Pdx‐1 in INS‐1 cells. Together these findings suggest that LNT could protect against pancreatic β‐cell apoptosis and dysfunction caused by STZ and therefore may be a potential pharmacological agent for preventing pancreatic β‐cell damage caused by oxidative stress associated with diabetes.     

N‐cadherin is dispensable for pancreas development but required for β‐cell granule turnover

The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N‐cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N‐cadherin during pancreas formation and function we generated a tissue‐specific knockout of N‐cadherin in the early pancreatic epithelium by inter‐crossing N‐cadherin‐floxed mice with Pdx1Cre mice. Analysis of pancreas‐specific ablation of N‐cadherin demonstrates that N‐cadherin is dispensable for pancreatic development, but required for β‐cell granule turnover. The number of insulin secretory granules is significantly reduced in N‐cadherin‐deficient β‐cells, and as a consequence insulin secretion is decreased. genesis 48:374–381, 2010. © 2010 Wiley‐Liss, Inc.     

Integrin β‐3 is required for the attachment,retention and therapeutic benefits of human cardiospheres in myocardial infarction

Cardiovascular diseases remain the leading causes of death worldwide. Stem cell therapy offers a promising option to regenerate injured myocardium. Among the various types of stem cells, cardiosphere cells represent a mixture of intrinsic heart stem cells and supporting cells. The safety and efficacy of cardiosphere cells have been demonstrated in recent clinical trials. Cell–matrix interaction plays an important role in mediating the engraftment of injected stem cells. Here, we studied the role of integrin β‐3 in cardiosphere‐mediated cell therapy in a mouse model of myocardial infarction. Our results indicated that inhibiting integrin β‐3 reduced attachment, retention and therapeutic benefits of human cardiospheres in mice with acute myocardial infarction. This suggests integrin β‐3 plays an important role in cardiosphere‐mediated heart regeneration.     

Integration of nano‐ and biotechnology for beta‐cell and islet transplantation in type‐1 diabetes treatment

Regenerative medicine using human or porcine β‐cells or islets has an excellent potential to become a clinically relevant method for the treatment of type‐1 diabetes. High‐resolution imaging of the function and faith of transplanted porcine pancreatic islets and human stem cell–derived beta cells in large animals and patients for testing advanced therapy medicinal products (ATMPs) is a currently unmet need for pre‐clinical/clinical trials. The iNanoBIT EU H2020 project is developing novel highly sensitive nanotechnology‐based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole‐body distribution of the cellular transplants in a porcine diabetes model with excellent translational potential to humans. We develop and validate the application of single‐photon emission computed tomography (SPECT) and optoacoustic imaging technologies in a transgenic insulin‐deficient pig model to observe transplanted porcine xeno‐islets and in vitro differentiated human beta cells. We are progressing in generating new transgenic reporter pigs and human‐induced pluripotent cell (iPSC) lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high‐resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.     

Impaired integrin‐mediated adhesion contributes to reduced barrier properties in VASP‐deficient microvascular endothelium

Recent studies point to a significant role of vasodilator‐stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions in vivo and in vitro. Moreover, it has been reported that VASP is required for activation of the small GTPase Rac 1. However, little is known whether VASP is involved in the regulation of cell adhesion molecules that are critical for maintenance of the endothelial barrier. Here we demonstrate that impaired barrier properties in VASP‐deficient (VASP?/?) microvascular myocardial endothelial cells (MyEnd) correlated with both impaired integrin‐mediated adhesion as revealed by laser tweezer trapping and reduced integrin‐dependent cell migration. This was paralleled by reduction of focal adhesions at the cell periphery as well as of β₁‐integrin and VE‐cadherin cytoskeletal anchorage. Incubation of MyEnd VASP wt with RGD peptide to block interaction of integrins with extracellular matrix (ECM) reduced barrier properties and Rac 1 activity in wt endothelial monolayers mimicking the situation in VASP (?/?) cells under resting conditions. Moreover, cAMP‐mediated Rac 1 activation was reduced under conditions of impaired integrin‐mediated adhesion in wt cells and cAMP‐induced increase in VE‐cadherin cytoskeletal anchorage was abolished in VASP (?/?) endothelium. In summary, these data indicate that VASP is required for integrin‐mediated adhesion which stabilizes endothelial barrier properties at least in part by facilitating Rac 1 activation. J. Cell. Physiol. 220: 357–366, 2009. © 2009 Wiley‐Liss, Inc.     

Thymus‐specific deletion of insulin induces autoimmune diabetes

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β‐cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire‐expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β‐cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID‐TEC mice for insulin‐deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β‐cell‐specific autoimmune destruction was observed, as well as islet‐specific T cell infiltration. The presence of insulin‐specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti‐insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes‐resistant HLA class II alleles. ID‐TEC mice could serve as a new model for studying this pathology.     

Integrin–TGF‐β crosstalk in fibrosis,cancer and wound healing

Accumulating evidence indicates that there is extensive crosstalk between integrins and TGF‐β signalling. TGF‐β affects integrin‐mediated cell adhesion and migration by regulating the expression of integrins, their ligands and integrin‐associated proteins. Conversely, several integrins directly control TGF‐β activation. In addition, a number of integrins can interfere with both Smad‐dependent and Smad‐independent TGF‐β signalling in different ways, including the regulation of the expression of TGF‐β signalling pathway components, the physical association of integrins with TGF‐β receptors and the modulation of downstream effectors. Reciprocal TGF‐β–integrin signalling is implicated in normal physiology, as well as in a variety of pathological processes including systemic sclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cancer; thus, integrins could provide attractive therapeutic targets to interfere with TGF‐β signalling in these processes.