Co-expression and regulation of connexins 36 and 43 in cultured neonatal rat pancreatic islets

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.     

Influence of Matricial Molecules on Growth and Differentiation of Entrapped Chondrocytes

Primary cultures of rabbit articular chondrocytes have been cultivated normally and within three-dimensional systems using different alginate matrices. The in vitro proliferation capacity of the cells immobilized in the calcium alginate beads was investigated. The growth curve showed that chondrocytes are able to grow and to divide for several days inside the beads; in parallel an increase in protein contents was also measured. The differentiated phenotype of rabbit articular chondrocytes consists of cartilage-specific proteoglycans. During serial monolayer cultures this phenotype was lost and replaced by a low level of proteoglycan synthesis. On the contrary when cultivated in beads, entrapped cells maintained their differentiated pheno-type over time; the rates of proteoglycan were similar to those of primary chondrocytes. All these parameters were tested comparatively using different substrata in monolayer cultures and in alginate gels. Assays were carried out to assess the influence of type I collagen, type IV collagen, and of fibronectine on the growth as well as on the differentiation phenotype. The encapsulation methodology is readily applicable to the culture of chondrocytes in single beads, in multiwell dishes, or to mass culture for a bioproduction of extracellular matrix components.     

The Effect of Corticosterone on the Growth of Perinatal Rat Lung Fibroblasts in Culture: Modulation by the Extracellular Matrix

Third passage lung fibroblasts from rat pups between the ages of fetal day 17 and postnatal day 15 were allowed to attach onto either tissue culture plastic or an endothelial cell-derived matrix, and were then exposed to different concentrations of corticosterone in culture medium containing 1% charcoal-stripped serum. The effect of the hormone on growth of these cells was assessed after 48 hours of exposure by radiothymidine incorporation into DNA. Lung fibroblasts on plastic responded to the hormone in an age-dependent manner; thus, cells which were obtained during late gestation (days 19–21) and after the third day of extrauterine life were consistently growth-inhibited by corticosterone, whereas those which were obtained from fetal donors prior to day 19 and from neonatal donors (days 0–3 after birth) were stimulated to grow in response to the hormone. On the other hand, cells that were plated onto an endothelial cell-derived matrix showed a different age-related response to the hormone. Thus, lung fibroblasts from all fetal donors and from postnatal donors up until 4–5 days of age were stimulated to grow in response to corticosterone. This modulatory effect of the matrix on the fibroblast response to corticosterone was also seen in early passage fibroblasts. In an attempt to identify the modulatory agent(s) in the extracellular matrix, fibroblasts from day 19 fetal rat lung were challenged with corticosterone in the presence of laminin, fibronectin, type IV collagen, type I collagen, heparin, or chondroitin sulfate. None of these agents exerted a modulatory effect resembling that seen with the extracellular matrix. These results suggest the existence during lung development of a fibroblast-endothelial interaction, the nature of which remains to be elucidated, which may serve to modify the effects of circulating hormones.     

Changing interactions between astrocytes and neurons during CNS maturation

The environments of the developing brain and injured adult brain differ in their abilities to support axonal growth. To determine if astrocytes contribute to this difference, neurons were plated onto astrocytes cultured from the neonatal rat cortex and from the injured adult brain. Two patterns of neurite growth were observed in these two astrocyte culture systems. Neurons contacting the neonatal astrocytes had neurites that were twice as long as those contacting the injured adult astrocytes. Furthermore, in cultures with neonatal astrocytes, neurites faithfully followed the astrocytic processes, maximizing their contact, while in cultures of injured adult astrocytes, the neurites had a tendency to cross the processes orthogonally, minimizing their interaction with the astrocytes. When neurons were grown suspended over either neonatal or injured adult astrocytes, no difference in neurite length or the pattern of neurite growth was observed, indicating that neurite growth was not differentially affected by soluble factors released from the two populations of astrocytes. The addition of fetal calf serum, which is known to contain protease inhibitors, did not alter neurite growth when compared to serum-free medium, suggesting that a substantial difference in protease activity does not account for the variations in neurite length observed. Based on these results, it appears that the molecular components of the external surface of injured adult astrocytes do not support neurite growth to the same extent as those found on neonatal astrocytes. The differing abilities of these two populations of cultured astrocytes to support neurite growth in culture may reflect a change in the functional role of these cells that occurs during the development of the central nervous system.     

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

Summary The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral micro-vasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

Comparison of expression of cathepsins B and L and MMP2 in endothelial cells and in capillary sprouting in collagen gel

The lysosomal cysteine proteinases cathepsins B and L are known to play an important role in the invasive growth of tumor cells, but their association with angiogenesis has been less well studied. The aim of this study was to determine the possible role of endothelial cell-associated cathepsins B and L in induced capillary growth in the aorta ring model of angiogenesis. Specific inhibitors of cysteine proteinases did not inhibit capillary growth in aorta ring culture and only slightly inhibited the degradation of surrounding collagen. In contrast, strong inhibition of both processes by the matrix metalloproteinase inhibitor BB-94 was observed, indicating the importance of endogenous MMP production in angiogenesis. In support of this finding, we demonstrated a significant increase in endogenous endothelial mRNA of MMP2, but not of cathepsins B and L, in proliferating primary human dermal microvascular endothelial cells (HMVEC-d) in culture. However, MMP2 mRNA expression was increased only when the cells were embedded in collagen but not when they were grown on plastic, regardless of the addition of the growth factors VEGF or bFGF. Moreover, on plastic the impairment of MMP2 induction by growth factors was observed. The differential effect of growth factors implies the crosstalk with integrin signaling as a consequence of binding to the different matrix. This study suggests that endothelial cell-associated cathepsins B and L are not involved in the invasive growth of capillaries from existing blood vessels and that the presence of collagen is necessary for MMP2 expression in endothelial cells.     

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.     

Growth factors upregulate deposition and remodeling of ECM by endothelial cells cultured for tissue-engineering applications

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial for their long-term function. The interaction between cells and extra-cellular components is indispensable in determining cellular behavior in tissues and on biomaterials. The fibrin that contains fibronectin shows promise in most aspects as a tissue engineering scaffold, whereas, deposition of elastin and collagen by endothelial cells grown in the lumen of the construct is desirable to improve post implant retention, mechanical stability and vaso-responsiveness. So far there is no report on production of extra-cellular matrix (ECM) proteins, elastin and collagen by endothelial cells (EC) in in vitro culture conditions. In this study, we have used a biomimetic approach of providing multiple growth factors (GF) in the fibronectin (FN)-containing fibrin matrix to induce production of elastin and collagen by the endothelial cells for application in vascular tissue engineering. Deposition of elastin and collagens with matrix remodeling is demonstrated through qualitative analysis of the matrices that were recovered after growing cells on the initial fibrin-FN-GF matrix. Expressions of mRNA for both proteins were assessed by real time polymerase chain reaction (RT-PCR) to estimate the effects of multiple growth factor compositions. Marked deposition of elastin and collagen was evidenced by staining the recovered matrix after different culture intervals. Obviously, the biomimetic environment created by adding angiogenic and platelet growth factors in the fibrin-fibronectin-gelatin matrix can induce deposition of collagens and elastin by EC.     

Primate embryonic stem cells create their own niche while differentiating in three-dimensional culture systems

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three-dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Effect of thawing rate and post-thaw culture on the cryopreserved fetal rat islets: functional and morphological correlation

The ability of the fetal pancreatic islet cells to multiply rendered them a potential tissue for transplantation studies to cure diabetes. A bank of fetal islets could be created with proper storage in liquid nitrogen. The aim of this study is to evaluate the effect of thawing rate and post-thaw culture on the structural and functional integrity of isolated cryopreserved islets of rat fetuses. Fetal rat islets were isolated by the collagenase digestion, cultured for three days, and then cryopreserved using dimethylsulphoxide as cryoprotectant and the step-rate cooling to -40 degrees C before immersing them in liquid nitrogen. The islets were thawed by the slow or fast warming rates using hyperosmolar sucrose solution and then cultured for 1 or 2 days. Insulin and C-peptide contents of the slow thawed islets were higher than those of the control. In the fast thawed islets the contents were similar to those of the control. Insulin and C-peptide release in response to glucose for the slow thawed islets were lower than those of the control and in the fast thawed islets they were similar to that of the control. Histological examination showed irregular periphery and fragmented central part of the large slowly thawed islets, which showed also variable immunohistochemical reaction to anti-insulin serum, ranging from strongly positive reaction to markedly weak reaction. Fast thawed islets showed mostly regular periphery and their reaction to the anti-insulin serum was slightly weaker than that of the control islets. It was concluded that fast thawing and post-thaw culture is much better than slow thawing, as indicated by nearly normal insulin and C-peptide content and release and intact structural integrity.     

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.     

Propagation of fetal human RPE cells: preservation of original culture morphology after serial passage

The permissive effects of extracellular matrix (ECM) on in vitro growth and differentiation of fetal human retinal pigment epithelial (RPE) cells have been studied. Factors which enhanced the effect of ECM to support cell division were also examined, including growth factors, culture media, and serum requirement. Under the specific culture conditions we have defined, it is possible to propagate these RPE cells at low density (less than 20 cells/mm2) with excellent growth properties for greater than 72 doublings (fourteen passages) in serial culture. Later-passaged cells maintained the morphological appearance of early-passaged cultures. ECM produced by bovine corneal endothelial cells was by far the most predominant factor in promoting rapid cell proliferation and viability over repeated passaging. Basic fibroblast growth factor (bFGF) exerted a substantial effect on the rate of cell division at different serum concentrations on plastic dishes. In addition, this factor showed profound synergistic effect when RPE cells were maintained on ECM, both in the preservation of cell morphology and also in long term viability. Other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-beta (TGF-B), were also tested, but EGF effects were less prominent than those observed with bFGF, and TGF-B had an inhibitory effect at high concentrations. The ability to obtain a relatively large number of human RPE cells in vitro which preserve the appearance of early passage cells may provide useful opportunities to study the physiological properties and pathological alterations involving this important cell type.     

Effects of nicotine on phenotypic modulation and initiation of DNA synthesis in cultured arterial smooth muscle cells

During the early stages of atherogenesis, as well as during in vitro cultivation, smooth muscle cells modulate from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex; it leads to decreased contractility and the commencement of cell growth and secretion of extracellular matrix components. In this paper, the effects of nicotine on adult rat arterial smooth muscle cells cultivated in vitro were studied by transmission electron microscopy and 3H-thymidine autoradiography. The results show that the drug speeded the initial rate of transition of the cells from contractile to synthetic phenotype in primary culture. Further, it stimulated the initiation of DNA synthesis in growth-arrested secondary cultures. Its effect was independent of other mitogens and additive to that of serum. The influences of nicotine, both on the modulation of the smooth muscle phenotype and the initiation of DNA synthesis, occurred at concentrations lower than those obtained in the blood after smoking and could contribute to the role of smoking as a risk factor for atherosclerosis.     

Chemotactic activity of culture supernatants of free and encapsulated pancreatic rat islets towards peritoneal macrophages.

Longterm efficiency of encapsulated pancreatic islet transplantation is limited by macrophagic reaction at the surface of biocompatible membrane. The aim of this work was to investigate the influence of soluble factors released by free and encapsulated islets on macrophage chemotaxis. The culture mediums conditioned for 6 days by free and encapsulated rat islets were incubated with peritoneal murine, rat allo and syngenic macrophages to study their migration. Culture supernatants of rat fibroblasts and acinar cells, glucose-stimulated free rat islets and supernatants of free rat islets treated by heat and proteinase K were also tested for their chemotactic activity. Islets encapsulation decreased the chemotactic activity of culture medium conditioned for 6 days by free rat islets on murine (1.66 +/- 0.20 vs. 3.10 +/- 0.23; p < 0.001, n = 5) and rat allogenic macrophages (1.63 +/- 0.21 vs. 4.70 +/- 0.36; p < 0.001, n = 9). There was no migration of rat macrophages towards syngenic islets. Fibroblasts exhibited a very strong chemotactic effect as compared to acinar cells. Insulin was not involved in macrophage migration. Proteinase K treatment of culture supernatant of free rat islets totally inhibited the chemotactic activity. After heating at 56 degrees C and 100 degrees C, this activity was reduced to 41 +/- 7% and 32 +/- 5% of the initial activity, respectively. In conclusion, pancreatic islet stimulated macrophage migration by release of immunological specific proteins partly retained by macroencapsulation.     

Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids,triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells

The regulation of early fetal germ cell growth has not been studied in cell culture, probably due to the poor survival of these cells. However, cell culture is the only system in which the control of cell growth can be studied independently of the influence of secreted testicular factors, which are diluted in the medium. We successfully cultured dispersed testicular cells from 16.5-day-old rat fetuses in defined medium and compared the growth of these cells with that of cells from 3-day-old neonates. In this system, fetal gonocytes displayed low levels of mitotic activity and their numbers remained stable. In contrast, neonatal gonocytes displayed high levels of mitotic activity and increased in number, these characteristics resembling those observed in vivo. We found that retinoic acid had deleterious effects on the number of gonocytes but did not affect Sertoli cell proliferation in fetal and neonatal cell cultures. Moreover, in fetal cell cultures, the decrease in the number of gonocytes resulted from a decrease in mitotic activity, probably due to a direct effect of retinoids on fetal gonocytes. Among the selective agonists for the retinoic acid receptor (RARalpha agonist, RARbeta agonist, and RARgamma agonist) and the retinoic X receptor (pan-RXR agonist) tested, only the RARalpha agonist reproduced the effects of retinoic acid at concentrations lower than its Kd value in both fetal and neonatal cell cultures. As both RARalpha and RXRalpha are present in fetal and neonatal gonocytes, we suggest that retinoic acid exerts its effects on gonocytes via a RARalpha-RXRalpha heterodimer, with RARalpha functioning as an active partner and RXRalpha as a passive partner. In this culture system, we show for the first time that triiodothyronine (T3) inhibits testicular fetal Sertoli cell and germ cell growth. We also tested intracellular signaling factors and found that a cAMP analog increased Sertoli cell proliferation and germ cell survival in both fetal and neonatal cells whereas phorbol esters (PMA) strongly inhibited the proliferation of fetal but not of neonatal gonocytes. None of the tested factors (T3, dbcAMP, and PMA) seemed to interact with the all-trans retinoic acid pathway. Thus, fetal gonocytes and neonatal gonocytes differ in intrinsic properties, and their growth is not regulated in the same manner. Despite their low level of mitotic activity, fetal gonocytes were more sensitive to various factors than neonatal gonocytes.     

Engineered vascular beds provide key signals to pancreatic hormone-producing cells

The mechanisms underlying early islet graft failure are not entirely clear, but are thought to involve ischemic injury due to delayed vascularization. We hypothesize that blood vessels play an active role in cell-cell communications supporting islet survival and engraftment. To test this hypothesis and to uncouple endothelial cell (EC)-generated signaling stimuli from their nutritional and gas exchange functions, we developed three dimensional (3D) endothelial vessel networks in engineered pancreatic tissues prepared from islets, fibroblasts and ECs. The tri-culture setup, seeded on highly porous biocompatible polymeric scaffolds closely mimics the natural anatomical context of pancreatic vasculature. Enhanced islet survival correlating with formation of functional tube-like endothelial vessels was demonstrated. Addition of foreskin fibroblasts to islet-endothelial cultures promoted tube-like structure formation, which further supported islet survival as well as insulin secretion. Gene expression profiles of EC growth factors, extracellular matrix (ECM), morphogenes and differentiation markers were significantly different in 2D versus 3D culture systems and were further modified upon addition of fibroblasts. Implantation of prevascularized islets into diabetic mice promoted survival, integration and function of the engrafted engineered tissue, supporting the suggested role of ECs in islet survival. These findings present potential strategies for preparation of transplantable islets with increased survival prospects.     

INGAP-related pentadecapeptide: its modulatory effect upon insulin secretion

We examined the effects of a pentadecapeptide having the 104-118 aminoacid sequence of islet neogenesis-associated protein (INGAP-PP) on insulin secretion, and the morphological characteristics of adult and neonatal pancreatic rat islets cultured in RPMI and 10 mM glucose for 4 days, with or without different INGAP-PP concentrations (0.1-100 mug/ml). A scrambled 15 aminoacid peptide was used as control for the specificity of INGAP-PP effect. Cultured neonatal and adult islets released insulin in response to glucose (2.8-16.7 mM) in a dose-dependent manner, and to leucine and arginine (10 mM). In all cases, the response was greater in adult islets. INGAP-PP added to the culture medium significantly enhanced glucose- and aminoacid-induced insulin release in both adult and newborn rats; however, no changes were observed with the scrambled peptide. Similar results were obtained incubating freshly isolated adult rat islets with INGAP-PP. Whereas INGAP-PP did not induce significant changes in islet survival rate or proportion/number of islet cells, it increased significantly beta-cell size. This first demonstration of the enhancing effect of INGAP-PP on the beta-cell secretory response of adult and newborn islets opens a new avenue to study its production mechanism and potential use to increase the secretory capacity of endogenous islets in intact animals or of islets preserved for future transplants.     

Metabolite control of angiogenesis: angiogenic effect of citrate

Endothelial cells respond to hypoxic changes with resultant accumulation of several metabolites and switch over to angiogenic phenotype. Although certain intermediates of glycolytic and oxidative metabolic pathways have been known to affect angiogenesis, the effect of citrate, which accumulates in certain tumors, on angiogenesis is not known. Therefore, the effect of citrate on angiogenesis was studied using different model systems. Increased vascularization in chorioallantoic membrane assay, increased endothelial sprouting in rat aortic rings, and increased expression of CD31, E-selectin in endothelial cells suggested a possible proangiogenic effect of citrate. Upregulation of angiogenic factors such as vascular endothelial growth factor and fibroblast growth factor suggested that the effect of citrate involves modulation of expression of angiogenic growth factors. LY 294002, an inhibitor of PI3K–Akt pathway, and wortmannin, an inhibitor of Akt pathway, reversed the effect of citrate in human umbilical vein endothelial cells. Citrate induced significant upregulation and activation of Akt in endothelial cells. Rapamycin, an inhibitor of mTOR, also reversed the effect of citrate in human umbilical vein endothelial cells and sprouting of aortic rings suggesting that the angiogenic effect of citrate involves activation of PI3K–Akt–mTOR pathway.