Papers by Vincenzo Cirulli
iScience, Sep 1, 2019
HIGHLIGHTS Cx43 is upregulated during hESC differentiation into definitive endoderm (DE) siRNA-me... more HIGHLIGHTS Cx43 is upregulated during hESC differentiation into definitive endoderm (DE) siRNA-mediated knockdown of Cx43 prevents hESC differentiation into DE cells AAP10-mediated gating of Cx43 gap junctions promotes the expression of FoxA2 and Sox17 Cx43 gap junction communication supports DE and pancreatic progenitors development
The FASEB Journal, Apr 1, 2010
Scientific Reports, Dec 16, 2016
Functional characterization of individual cells within heterogeneous tissue preparations is chall... more Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H 2 O 2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type. A need for functional assessment of heterogeneous mixture of cells A common challenge in cell biology is the need to assess the functional attributes of isolated primary cells in heterogeneous cell mixtures. One example involves studies of directed differentiation of stem cells toward a given cell type of interest. Differences in cell fate specification, inefficient transitions of a given cell phenotype through specific stages of development, and intrinsic heterogeneity existing within populations of progenitor cells 1 can each result in complex admixtures of many distinct cell types, and identifying and characterizing individual cell types in that mixture can be challenging. Other examples include the need to identify and characterize cells isolated from primary tissues such as liver 2,3 , pancreatic islets 4,5 , brain 6 , cardiomyocytes 7 or blood leukocytes 8. Assessing cellular differences in drug toxicity within a given tissue preparation can also be confounded if, for example, a sparsely represented cell type, but not the major parenchymal cell type, is targeted and eliminated by the drug. The ability to discriminate between these selective drug effects requires high-throughput cellular analysis methods that are not currently available. These examples highlight instances in which measures of bulk cell response are uninformative with respect to cell-specific behavior. Even homogeneous cell mixtures can be characterized by wide variability in individual cellular responses, the nature of which may be physiologically or pathophysiologically important to characterize 9. Such challenges can be addressed through an approach to single cell functional assessment that permits statistical analysis of the distributions of the responses. Achieving this goal, however, requires either that the cells are
Frontiers in Physiology, Aug 31, 2016
Fibrotic disorders involve replacement of normal parenchyma with myofibroblasts, which deposit co... more Fibrotic disorders involve replacement of normal parenchyma with myofibroblasts, which deposit connective tissue, leading to obliteration of the function of the underlying organ. The treatment options are inadequate and reflect the fact that signaling targets in myofibroblasts are unknown. Here we identify the hyperactive Lyn signaling in myofibroblasts of patients with chronic pancreatitis-induced fibrosis. Lyn activation coexpress with markers of activated myofibroblasts, and is increased ∼11-fold in chronic pancreatitis compared to normal tissue. Inhibition of Lyn with siRNA or INNO-406 leads to the substantial decrease of migration and proliferation of human chronic pancreatitis myofibroblasts in vitro, while leaving migration and proliferation of normal myofibroblasts only slightly affected. Furthermore, inhibition of Lyn prevents synthesis of procollagen and collagen in myofibroblasts in a mouse model of chronic pancreatitis-induced fibrosis. We conclude that Lyn, as a positive regulator of myofibroblast migration, proliferation, and collagen production, is a key target for preventing fibrosis.
Biology of the Cell, Nov 1, 2002
Connexin channels clustered at gap junctions are obligatory attributes of all macroscopic endocri... more Connexin channels clustered at gap junctions are obligatory attributes of all macroscopic endocrine and exocrine glands investigated so far and also connect most types of cells which produce secretory products in other tissues. Increasing evidence indicates that connexins, and the cell-to-cell communications that these proteins permit, contribute to control the growth of secretory cells, their expression of specific genes and their differentiated function, including their characteristic ability to biosynthetize and release secretory products in a regulated manner. Since the previous reviews which have been published on this topic, several lines of evidence have been added in support of multiple regulatory roles of gland connexins. Here, we review this novel evidence, point to the many questions which are still open and discuss some interesting perspectives of the field.
Cell Calcium, Dec 1, 2004
Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca 2+ sign... more Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca 2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca 2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca 2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca 2+ ] c), mitochondria ([Ca 2+ ] m) and ER ([Ca 2+ ] ER) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca 2+ ] were unaffected. By contrast, Ca 2+ influx elicited significantly smaller increases in [Ca 2+ ] c and [Ca 2+ ] m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca 2+ influx, but that the transfer of Ca 2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles.
Journal of Biological Chemistry, Jul 1, 2004
The cell adhesion molecule L1 has been implicated in a variety of motile processes, including neu... more The cell adhesion molecule L1 has been implicated in a variety of motile processes, including neurite extension, cerebellar cell migration, extravasation, and metastasis. Homophilic or heterophilic L1 binding and concomitant signaling have been shown to promote cell motility in the short term. In this report, L1 is also shown to induce and maintain a motile and invasive phenotype by promoting gene transcription. In the presence of serum or platelet-derived growth factor, L1 promotes heightened and sustained activation of the extracellular signal-regulated kinase pathway. Activation of this pathway then induces the expression of motilityand invasion-associated gene products, including the  3-integrin subunit, small GTPases, and the cysteine proteases cathepsin-L and-B. Induction of integrin ␣ v  3 and rac-1 is shown to contribute directly to L1-dependent haptotaxis, whereas induction of cathepsins-L and-B promotes matrix invasion. This study provides a novel translational mechanism to account for the association between L1 expression and motile processes involved in metastasis and development.
Nature Reviews Molecular Cell Biology, Mar 14, 2007
The EMBO Journal, Nov 28, 2012
Cell Reports, Aug 1, 2017
The development and function of epithelia depend on the establishment and maintenance of cellcell... more The development and function of epithelia depend on the establishment and maintenance of cellcell adhesion and intercellular junctions, which operate as mechanosensor hubs for the transduction of biochemical signals regulating cell proliferation, differentiation, survival, and regeneration. Here, we show that αE-catenin, a key component of adherens junctions, functions as a positive regulator of pancreatic islet cell lineage differentiation by repressing the sonic hedgehog pathway (SHH). Thus, deletion of αE-catenin in multipotent pancreatic progenitors resulted in (1) loss of adherens junctions, (2) constitutive activation of SHH, (3) decrease in islet cell lineage differentiation, and (4) accumulation of immature Sox9 + progenitors. Pharmacological blockade of SHH signaling in pancreatic organ cultures and in vivo rescued this defect, allowing αE-cateninnull Sox9 + pancreatic progenitors to differentiate into endocrine cells. The results uncover crucial functions of αE-catenin in pancreatic islet development and harbor significant implications for the design of β cell replacement and regeneration therapies in diabetes.
Journal of Clinical Investigation, Jun 1, 1991
To determine whether pancreatic B cells show a constant secretion pattern during repeated stimula... more To determine whether pancreatic B cells show a constant secretion pattern during repeated stimulations, we have used a sequential hemolytic plaque assay to monitor their individual insulin release during several successive 30-min incubations in the presence of 16.7 mM glucose. We have found that the total B cell secretion did not vary significantly in these successive glucose stimulations and that, under these conditions, the majority of B cells that were stimulated to release insulin during the first incubation also secreted during the second, third, and, when this was tested, during the fourth incubation. Similarly, most of the B cells that did not release detectable amounts of insulin during the first incubation did not secrete also during the two (or three) subsequent secretion tests. Together, the two groups of B cells that showed a constant secretory pattern, represented-75% of the entire B cell population. The remaining 25% of B cells shifted from a secreting to a non-secreting state, or vice versa, from one incubation to another. These observations were made under three different time frames in which we tested single B cells as well as B cell clusters at rather different intervals. These findings support the existence of distinct B cell subpopulations differing lastingly in their ability to secrete insulin in response to glucose.
Cell Reports, Nov 1, 2022
Human Molecular Genetics, Nov 10, 2008
Previous studies have documented that the insulin-producing b-cells of laboratory rodents are cou... more Previous studies have documented that the insulin-producing b-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes b-cells, leading to secretory defects reminiscent of those observed in type 2 diabetes. Since human islets differ in several respects from those of laboratory rodents, we have now screened human pancreas, and islets isolated thereof, for expression of a variety of connexin genes, tested whether the cognate proteins form functional channels for islet cell exchanges, and assessed whether this expression changes with b-cell function in islets of control and type 2 diabetics. Here, we show that (i) different connexin isoforms are differentially distributed in the exocrine and endocrine parts of the human pancreas; (ii) human islets express at the transcript level different connexin isoforms; (iii) the membrane of b-cells harbors detectable levels of gap junctions made of Cx36; (iv) this protein is concentrated in lipid raft domains of the b-cell membrane where it forms gap junctions; (v) Cx36 channels allow for the preferential exchange of cationic molecules between human b-cells; (vi) the levels of Cx36 mRNA correlated with the expression of the insulin gene in the islets of both control and type 2 diabetics. The data show that Cx36 is a native protein of human pancreatic islets, which mediates the coupling of the insulin-producing b-cells, and contributes to control b-cell function by modulating gene expression.
Diabetologia, Apr 1, 1996
Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pan... more Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells. [Diabetologia (1996) 39: 474-480] Keywords ICA69, pancreatic islets, antigen, insulindependent diabetes mellitus, pathogenesis, fetal development.
Journal of Biological Chemistry, Apr 1, 2004
The role of individual integrins in human -cell development and function is largely unknown. Thi... more The role of individual integrins in human -cell development and function is largely unknown. This study describes the contribution of ␣ v-integrins to human -cell adhesion, spreading, and motility. Developmental differences in ␣ v-integrin utilization are addressed by comparing the responses of adult and fetal -cells, and vitronectin is used as a substrate based on its unique pattern of expression in the developing pancreas. Fetal and adult -cells attached equally to vitronectin and integrin ␣ v  5 was found to support the adhesion of both mature and immature -cell populations. Fetal -cells were also observed to spread and migrate on vitronectin, and integrin ␣ v  1 was found to be essential for these responses. In contrast to their fetal counterparts, adult -cells failed to either spread or migrate and this deficit was associated with a marked down-regulation of ␣ v  1 expression in adult islet preparations. The integrin ␣ v  3 was not found to support significant -cell attachment or migration. Based on our findings, we conclude that integrins ␣ v  5 and ␣ v  1 are important mediators of human -cell adhesion and motility, respectively. By supporting fetal -cell migration, ␣ v  1 could play an important role in early motile processes required for islet neogenesis.
Journal of Cellular Physiology, 2010
A critical shortage of donor pancreata currently prevents the development of a universal cell-bas... more 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 b-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 b-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 b 1-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 b-cells undergoing mesenchymal transition were found to acquire a 1 b 1 expression, and ligation of this integrin then promotes declines in insulin gene expression and a marked increase in b-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 b-cell expansion protocols.
Journal of Clinical Investigation, May 1, 1993
The characteristic three-dimensional cell type organization of islets of Langerhans is perturbed ... more The characteristic three-dimensional cell type organization of islets of Langerhans is perturbed in animal models of diabetes, suggesting that it may be important for islet function. Rat islet cells in culture are able to form aggregates with an architecture similar to native islets (pseudoislets), thus providing a good model to study the molecular basis of islet architecture and its role in islet function. Sorted islet B cells and non-B cells were permanently labeled with two different fluorescent dyes (DiO and DiI), mixed, and allowed to form aggregates during a 5-d culture in the presence or absence of TNF-a (100 U/ml), a cytokine suggested to be implicated in the early physiological events leading to insulin-dependent diabetes mellitus. Confocal microscopy of aggregates revealed that TNF-a reversibly perturbs the typical segregation between B and non-B cells. Insulin secretion, was altered in the disorganized aggregates, and returned towards normal when pseudoislets had regained their typical architecture. The homotypic adhesion properties of sorted B and non-B cells cultured for 20 h in the presence or absence of TNF-a were studied in a short term aggregation assay. TNF-a induced a significant rise in Ca2"-independent adhesion of B cells (from 24±1.1% to 44.3+1.2%; n = 4, P <0.001). These findings raise the possibility that the increased expression of Ca2"-independent adhesion molecules on B cells leads to altered islet architecture, which might be a factor in the perturbation of islet function induced by TNF-a.
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Papers by Vincenzo Cirulli