Hypoxia induction in cultured pancreatic islets enhances endothelial cell morphology and survival while maintaining beta-cell function.

BACKGROUND: Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue. We now aim to determine the impact of hypoxia on islet vascularization. METHODS: We induced hypoxia in cultured mouse islets using the hypoxia mimetic cobalt chloride (100 µM CoCl2). We measured the impact on islet metabolism (two-photon NAD(P)H and Rh123 imaging) and function (insulin secretion and survival). We also measured the impact on hypoxia related transcripts (HIF-1α, VEGF-A, PDK-1, LDHA, COX4) and confirmed increased VEGF-A expression and secretion. Finally, we measured the vascularization of islets in static and flowing culture using PECAM-1 immunofluorescence. RESULTS: CoCl2 did not induce significant changes in beta cell metabolism (NAD(P)H and Rh123), insulin secretion, and survival. Consistent with hypoxia induction, CoCl2 stimulated HIF-1α, PDK-1, and LDHA transcripts and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2) and this switch was noted in the glucose-stimulated cytoplasmic NAD(P)H responses. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static (dish) and microfluidic flow culture. CONCLUSIONS: Hypoxia induction using CoCl2 had a positive effect on islet EC morphology and survival with limited impact on beta-cell metabolism, function, and survival. The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin.

A microfluidic device designed to induce media flow throughout pancreatic islets while limiting shear-induced damage.

Pancreatic islets are heavily vascularized in vivo with fenestrated endothelial cells (ECs) to facilitate blood glucose-sensing and endocrine hormone secretion. The close proximity of insulin secreting beta cells and ECs also plays a critical role in modulating the proliferation and survival of both cell types with the mechanisms governing this interaction poorly understood. Isolated islets lose EC morphology and mass over a period of days in culture prohibiting study of this interaction in vitro. The loss of ECs also limits the efficacy of islet transplant revascularization in the treatment of Type 1 diabetes. We previously showed that microfluidically driven flow positively affects beta-cell function and EC survival in culture due to enhanced transport of media into the tissue. However, holding islets stationary in media flow using a dam-wall design also resulted in reduced glucose-stimulated metabolic and Ca(2+) responses at the periphery of the tissue consistent with shear-induced damage. We have now created a device that traps islets into sequential cup-shaped nozzles. This hydrodynamic trap design limits flow velocity around the perimeter of the islet while enhancing media flow through the tissue. We demonstrate the feasibility of this device to dynamically treat and collect effluent from islets. We further show that treating islets in this device enhances EC morphology without reducing glucose-stimulate Ca(2+) responses. These data reveal a microfluidic device to study EC and endocrine cell interaction that can be further leveraged to prime islets prior to transplantation.

Fibroblast growth factor receptor like-1 (FGFRL1) interacts with SHP-1 phosphatase at insulin secretory granules and induces beta-cell ERK1/2 protein activation.

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and ßTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif.

Quantitative imaging of electron transfer flavoprotein autofluorescence reveals the dynamics of lipid partitioning in living pancreatic islets.

Pancreatic islet ß-cells metabolically sense nutrients to maintain blood glucose homeostasis through the regulated secretion of insulin. Long-term exposure to a mixed supply of excess glucose and fatty acids induces ß-cell dysfunction and type II diabetes in a process termed glucolipotoxicity. Despite a number of documented mechanisms for glucolipotoxicity, the interplay between glucose and fatty acid oxidation in islets remains debated. Here, we develop confocal imaging of electron transfer flavoprotein (ETF) autofluorescence to reveal the dynamics of fatty acid oxidation in living pancreatic islets. This method further integrates microfluidic devices to hold the islets stationary in flow, and thus achieve ETF imaging in the ß-cells with high spatial and temporal resolution. Our data first confirm that ETF autofluorescence reflects electron transport chain (ETC) activity downstream of Complex I, consistent with a response directly related to fatty acid metabolism. Together with two-photon imaging of NAD(P)H and confocal imaging of lipoamide dehydrogenase (LipDH) autofluorescence, we show that the ETC predominantly draws electrons from LipDH/NADH-dependent Complex I rather than from ETF/FADH(2)-dependent ETF:CoQ oxidoreductase (ETF-QO). Islets stimulated with palmitate also show increased ETF redox state that is dose-dependently diminished by glucose (>10 mM). Furthermore, stimulation with a glucose bolus causes a two-tier drop in the ETF redox state at ∼5 and ∼20 min, suggesting glucose metabolism immediately increases ETC activity and later decreases fatty acid oxidation. Our results demonstrate the utility of ETF imaging in characterizing fatty acid-induced redox responses with high subcellular and temporal resolution. Our results further demonstrate a dominant role of glucose metabolism over fatty acid oxidation in ß-cells even when presented with a mixed nutrient condition associated with glucolipotoxicity.

Dynamics and Distribution of Klothoß (KLB) and fibroblast growth factor receptor-1 (FGFR1) in living cells reveal the fibroblast growth factor-21 (FGF21)-induced receptor complex.

FGF21 stimulates FGFR1c activity in cells that co-express Klothoß (KLB); however, relatively little is known about the interaction of these receptors at the plasma membrane. We measured the dynamics and distribution of fluorescent protein-tagged KLB and FGFR1c in living cells using fluorescence recovery after photobleaching and number and brightness analysis. We confirmed that fluorescent protein-tagged KLB translocates to the plasma membrane and is active when co-expressed with FGFR1c. FGF21-induced signaling was enhanced in cells treated with lactose, a competitive inhibitor of the galectin lattice, suggesting that lattice-binding modulates KLB and/or FGFR1c activity. Fluorescence recovery after photobleaching analysis consistently revealed that lactose treatment increased KLB mobility at the plasma membrane, but did not affect the mobility of FGFR1c. The association of endogenous KLB with the galectin lattice was also confirmed by co-immunoprecipitation with galectin-3. KLB mobility increased when co-expressed with FGFR1c, suggesting that the two receptors form a heterocomplex independent of the galectin lattice. Number and brightness analysis revealed that KLB and FGFR1c behave as monomers and dimers at the plasma membrane, respectively. Co-expression resulted in monomeric expression of KLB and FGFR1c consistent with formation of a 1:1 heterocomplex. Subsequent addition of FGF21 induced FGFR1 dimerization without changing KLB aggregate size, suggesting formation of a 1:2 KLB-FGFR1c signaling complex. Overall, these data suggest that KLB and FGFR1 form a 1:1 heterocomplex independent of the galectin lattice that transitions to a 1:2 complex upon the addition of FGF21.

Autofluorescence imaging of living pancreatic islets reveals fibroblast growth factor-21 (FGF21)-induced metabolism.

Fibroblast growth factor-21 (FGF21) has therapeutic potential for metabolic syndrome due to positive effects on fatty acid metabolism in liver and white adipose tissue. FGF21 also improves pancreatic islet survival in excess palmitate; however, much less is known about FGF21-induced metabolism in this tissue. We first confirmed FGF21-dependent activity in islets by identifying expression of the cognate coreceptor Klothoß, and by measuring a ligand-stimulated decrease in acetyl-CoA carboxylase expression. To further reveal the effect of FGF21 on metabolism, we employed a unique combination of two-photon and confocal autofluorescence imaging of the NAD(P)H and mitochondrial NADH responses while holding living islets stationary in a microfluidic device. These responses were further correlated to mitochondrial membrane potential and insulin secretion. Glucose-stimulated responses were relatively unchanged by FGF21. In contrast, responses to glucose in the presence of palmitate were significantly reduced compared to controls showing diminished NAD(P)H, mitochondrial NADH, mitochondrial membrane potential, and insulin secretion. Consistent with the glucose-stimulated responses being smaller due to continued fatty acid oxidation, mitochondrial membrane potential was increased in FGF21-treated islets by using the fatty acid transport inhibitor etomoxir. Citrate-stimulated NADPH responses were also significantly larger in FGF21-treated islets suggesting preference for citrate cycling rather than acetyl-CoA carboxylase-dependent fatty acid synthesis. Overall, these data show a reduction in palmitate-induced potentiation of glucose-stimulated metabolism and insulin secretion in FGF21-treated islets, and establish the use of autofluorescence imaging and microfluidic devices to investigate cell metabolism in a limited amount of living tissue.

Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells.

Pancreatic islets are heavily vascularized in vivo with each insulin secreting beta-cell associated with at least one endothelial cell (EC). This structure is maintained immediately post-isolation; however, in culture the ECs slowly deteriorate, losing density and branched morphology. We postulate that this deterioration occurs in the absence of blood flow due to limited diffusion of media inside the tissue. To improve exchange of media inside the tissue, we created a microfluidic device to culture islets in a range of flow-rates. Culturing the islets from C57BL6 mice in this device with media flowing between 1 and 7 ml/24 hr resulted in twice the EC-density and -connected length compared to classically cultured islets. Media containing fluorescent dextran reached the center of islets in the device in a flow-rate-dependant manner consistent with improved penetration. We also observed deterioration of EC morphology using serum free media that was rescued by addition of bovine serum albumin, a known anti-apoptotic signal with limited diffusion in tissue. We further examined the effect of flow on beta-cells showing dampened glucose-stimulated Ca(2+)-response from cells at the periphery of the islet where fluid shear-stress is greatest. However, we observed normal two-photon NAD(P)H response and insulin secretion from the remainder of the islet. These data reveal the deterioration of islet EC-morphology is in part due to restricted diffusion of serum albumin within the tissue. These data further reveal microfluidic devices as unique platforms to optimize islet culture by introducing intercellular flow to overcome the restricted diffusion of media components.

Altered expression of disintegrin metalloproteinases and their inhibitor in human dilated cardiomyopathy.

BACKGROUND: Disintegrin metalloproteinases (ADAMs) may contribute to structural cardiac remodeling by altering cell-surface matrix receptors (integrins) and activating potent biomolecules. We compared expression of ADAMs, their endogenous inhibitor tissue inhibitor of metalloproteinases (TIMP)-3, and integrins in human heart tissue with varied patterns of structural remodeling. METHODS AND RESULTS: Myocardium was obtained from patients with dilated cardiomyopathy (n=20), hypertrophic obstructive cardiomyopathy (n=5), and nonfailing donor hearts (n=7). Paired samples (n=10) were obtained before left ventricular assist device insertion and at transplantation. The expressions of ADAM10, ADAM12, ADAM15, and ADAM17, TIMP-3, and integrin receptors beta1D and beta3 were determined by quantitative immunoblotting. Integrin shedding was assessed by the ratio of integrin cleavage products to intact protein abundance. Confocal microscopy was performed. Dilated cardiomyopathy was characterized by increased ADAM10 and ADAM15 expression and reduced TIMP-3 expression. The integrin beta1D cleavage ratio was elevated, indicating receptor shedding. ADAM10 and ADAM15 expressions correlated with the cleavage ratio. ADAM10 colocalized with integrin beta1D by confocal microscopy. ADAM10 expression correlated with clinical indices of chamber dilatation and systolic dysfunction. Hemodynamic unloading reduced ADAM10 and ADAM12 expressions and increased integrin beta1D expression. ADAM12 and integrin beta1D expressions were increased in HOCM. ADAM17 was increased in both dilated cardiomyopathy and hypertrophic obstructive cardiomyopathy. CONCLUSIONS: Disintegrin metalloproteinases are differentially expressed in human myocardium, reflecting the underlying pattern of structural remodeling. ADAM10 and ADAM15 may contribute to cardiac dilatation by reducing cell-matrix interactions via integrin shedding. Targeting disintegrin metalloproteinases, perhaps by restoring deficient TIMP-3 levels with gene or cell-based therapies, may prevent progressive chamber dilatation in human dilated cardiomyopathy.

Cell transplantation preserves matrix homeostasis: a novel paracrine mechanism.

OBJECTIVES: Cell transplantation prevents chamber dilatation, but the underlying molecular mechanisms remain undefined. Structural cardiac remodeling involves matrix degradation from an imbalance of matrix metalloproteinases (MMP) relative to endogenous tissue inhibitors of metalloproteinases (TIMP). We aimed to determine the capacity of cell transplantation to alter extracellular matrix in the failing heart and, in so doing, identify novel paracrine molecular mediators underlying the beneficial effects of cell transplantation on chamber dilatation. METHODS: Smooth muscle cells were transplanted to the dilating left ventricle of cardiomyopathic hamsters (CTX, n = 15) compared with age-matched media-injected cardiomyopathic (CON, n = 15) and normal hamsters (n = 7). After 5 weeks, left ventricular volume was measured by computerized planimetry. Fibrillar collagen was examined by confocal microscopy. Matrix homeostasis was quantified by measuring MMP/TIMP expression/activity relative to myocardial collagen synthesis (14C-proline uptake). RESULTS: Left ventricular dilatation was attenuated in CTX hearts (P = .02). CTX restored perimysial collagen fiber content and architecture to normal levels. TIMP-2 and TIMP-3 expression were enhanced in CTX (TIMP-2, 195% +/- 42% of CON, P = .02; TIMP-3, 118% +/- 3% of CON, P = .002), and correspondingly, gelatinase MMP-2 activity was reduced (P < .05). The TIMP:MMP ratio was increased in CTX hearts (TIMP-2 to MMP-2, 410% +/- 134% of CON, P = .04, and TIMP-3 to MMP-9, 205% +/- 47% of CON, P = .03), reflecting a reduced capacity for matrix degradation. Collagen synthesis was equivalent (CTX vs CON), suggesting that restored matrix architecture was a function of attenuated matrix degradation. CONCLUSIONS: These data provide the first evidence that cell transplantation limits ventricular dilatation in the failing heart through a paracrine-mediated mechanism that preserves extracellular matrix homeostasis.

Matrix remodeling in experimental and human heart failure: a possible regulatory role for TIMP-3.

In the failing heart, an imbalance in matrix metalloproteinases (MMPs) and their biological regulators, the tissue inhibitors of MMPs (TIMPs), may result in cardiac dilatation from matrix degradation. We hypothesized that a reduction of myocardial TIMP-3 is associated with adverse matrix remodeling in both human and experimental heart failure. Cardiomyopathic hamsters at age 15 wk (normal), 25 wk (compensated stage), and 35 wk (overt failure) were compared with age-matched normal controls. MMP activity (gelatinase bioassay) was increased in cardiomyopathic hearts (P = 0.03) and peaked during the transition to overt heart failure. TIMP-3 content (immunoblot) was decreased compared with normal controls (74 +/- 5% at 25 wk, 69 +/- 10% at 35 wk; P = 0.001) and its reduction was associated with increased MMP activity (r = -0.6; P = 0.004). TIMP-1 increased progressively (P = 0.001), whereas TIMP-2, TIMP-4, and MMP protein levels were unchanged. Myocardial collagen (hydroxyproline content) increased with time during the progression to end-stage cardiac failure (P < 0.0001). Collagen synthesis ([(14)C]proline uptake) was elevated in cardiomyopathy at 15 and 25 wk (P < 0.05). The collagen cross-linking ratio (insoluble:soluble collagen) was reduced (P = 0.003) as the left ventricle dilated. By confocal microscopy restricted to viable myocardium, collagen content was reduced (P = 0.04) with fragmentation (P < 0.0001) and thinning (P = 0.003) of perimysial collagen fibers. Similarly, patients with end-stage congestive heart failure (n = 7) compared with nonfailing controls (n = 2) had elevated gelatinase MMP activity (P = 0.02) associated with isolated reductions in TIMP-3 (55 +/- 5% of normal; P = 0.003). Reductions of TIMP-3 parallel adverse matrix remodeling in the cardiomyopathic hamster and the failing human heart. TIMP-3 may contribute to the regulation of myocardial remodeling and its reduction may promote a transition from compensated to end-stage congestive heart failure.

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension.

An oxygen responsive transcription factor regulating human glutathione peroxidase gene (GPx) through two oxygen responsive elements (ORE I and ORE2) has been purified and characterized by sequence-specific DNA affinity chromatography. The DNA binding activity, termed Oxygen Responsive Element Binding Protein (OREBP), was partially represented by a 77 kD polypeptide (p70) possessing a blocked N-terminus. The p70 subunit co-eluted with an 86 kD subunit (p80) from affinity columns. N-terminal sequencing analysis of the 86 kD component revealed that this protein represented the larger member of the Ku antigen complex. The identity of the purified 77 kD subunit was determined by Western blot analysis using an antibody directed against the p70 protein. In addition to binding the GPx-ORE, the OREBP was itself regulated by oxygen tension. It was found that the abundance of the ORE binding activity was decreased in cells maintained at low oxygen tension (40 mm Hg). Anti-Ku-antibodies specifically supershifted the OREBP-ORE DNA complex. These observations further add to the numerous nuclear roles of the Ku-transcription factor.