The maternal-fetal transfer of passive immunity as a mechanism of transplacental nanoparticle drug delivery for prenatal therapies.

Nanoparticles administered into the maternal circulation and across the placenta are a potential clinical therapy to treat congenital diseases. The mechanism by which nanoparticles can safely cross the placenta for targeted drug delivery to the fetus remains poorly understood. We demonstrate that the maternal-fetal transfer of passive immunity through the neonatal Fc Receptor (FcRn) can induce the transplacental transfer of chitosan nanoparticles modifed with IgG antibodies (414 ± 27 nm). The transfer of FITC-tagged IgG-modified chitosan nanoparticles was 2.8 times higher (p = 0.0264) compared to similarly-sized unmodified chitosan nanoparticles (375 ± 17 nm). Co-administration of free IgG competitively diminished the transplacental transfer of IgG-modified nanoparticles, yet unmodified nanoparticles remained unaffected. Colocalization of the FcRn and the IgG-modified chitosan nanoparticles were observed with confocal microscopy. Barrier function before and after nanoparticle administration remained intact as determined by TEER (75-79 Ω cm2) and immmunofluorescence of ZO-1 tight junction proteins. The results provide insight into the clinical applications of nanoparticles for prenatal therapies using the mechanism of the maternal-fetal transfer of passive immunity.

Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3.

Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analog misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.

Oxidized phosphatidylcholines induce multiple functional defects in airway epithelial cells.

Oxidative stress is a hallmark of numerous airway diseases, contributing to extensive cell and tissue damage. Cell membranes and the airway mucosal lining are rich in phospholipids that are particularly susceptible to oxidative attack, producing bioactive molecules including oxidized phosphatidylcholines (OxPCs). With the recent discovery of elevated OxPCs in patients with asthma after allergen challenge, we hypothesized that OxPCs directly contribute to disease by inducing airway epithelial cell dysfunction. We found that OxPCs induced concentration-dependent cell stress and loss of viability in BEAS-2B and Calu-3 cell lines and primary human epithelial cells. These responses corresponded with significant epithelial barrier dysfunction, which was further compounded when combining OxPCs with an epithelial wound. OxPCs inhibited DNA synthesis and migration required to reestablish barrier function, but cells recovered if OxPCs were washed off soon after treatment. OxPCs induced generation of reactive oxygen species, lipid peroxidation, and mitochondrial dysfunction, raising the possibility that OxPCs cause pathological lipid metabolism in a self-propagating cycle. The oxidative stress induced by OxPCs could not be abrogated by putative OxPC receptor blockers, but partial recovery of barrier function, proliferation, and lipid peroxidation could be achieved with the antioxidant N-acetyl cysteine. In summary, we have identified OxPCs as a group of bioactive molecules that significantly impair multiple facets of epithelial cell function, consistent with pathological features of asthma. Further characterization of the mechanisms by which OxPCs affect epithelial cells could yield new insights into how oxidative stress contributes to the pathogenesis of airway disease.

BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance.Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3ß: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.

Misoprostol attenuates neonatal cardiomyocyte proliferation through Bnip3, perinuclear calcium signaling, and inhibition of glycolysis.

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

Correction to: Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition.

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.

Airway smooth muscle in asthma: linking contraction and mechanotransduction to disease pathogenesis and remodelling.

Asthma is an obstructive airway disease, with a heterogeneous and multifactorial pathogenesis. Although generally considered to be a disease principally driven by chronic inflammation, it is becoming increasingly recognised that the immune component of the pathology poorly correlates with the clinical symptoms of asthma, thus highlighting a potentially central role for non-immune cells. In this context airway smooth muscle (ASM) may be a key player, as it comprises a significant proportion of the airway wall and is the ultimate effector of acute airway narrowing. Historically, the contribution of ASM to asthma pathogenesis has been contentious, yet emerging evidence suggests that ASM contractile activation imparts chronic effects that extend well beyond the temporary effects of bronchoconstriction. In this review article we describe the effects that ASM contraction, in combination with cellular mechanotransduction and novel contraction-inflammation synergies, contribute to asthma pathogenesis. Specific emphasis will be placed on the effects that ASM contraction exerts on the mechanical properties of the airway wall, as well as novel mechanisms by which ASM contraction may contribute to more established features of asthma such as airway wall remodelling.

Models to study airway smooth muscle contraction in vivo, ex vivo and in vitro: implications in understanding asthma.

Asthma is a chronic obstructive airway disease characterised by airway hyperresponsiveness (AHR) and airway wall remodelling. The effector of airway narrowing is the contraction of airway smooth muscle (ASM), yet the question of whether an inherent or acquired dysfunction in ASM contractile function plays a significant role in the disease pathophysiology remains contentious. The difficulty in determining the role of ASM lies in limitations with the models used to assess contraction. In vivo models provide a fully integrated physiological response but ASM contraction cannot be directly measured. Ex vivo and in vitro models can provide more direct assessment of ASM contraction but the loss of factors that may modulate ASM responsiveness and AHR, including interaction between multiple cell types and disruption of the mechanical environment, precludes a complete understanding of the disease process. In this review we detail key advantages of common in vivo, ex vivo and in vitro models of ASM contraction, as well as emerging tissue engineered models of ASM and whole airways. We also highlight important findings from each model with respect to the pathophysiology of asthma.

Airway contractility and remodeling: links to asthma symptoms.

Respiratory symptoms are largely caused by obstruction of the airways. In asthma, airway narrowing mediated by airway smooth muscle (ASM) contraction contributes significantly to obstruction. The spasmogens produced following exposure to environmental triggers, such as viruses or allergens, are initially responsible for ASM activation. However, the extent of narrowing of the airway lumen due to ASM shortening can be influenced by many factors and it remains a real challenge to decipher the exact role of ASM in causing asthmatic symptoms. Innovative tools, such as the forced oscillation technique, continue to develop and have been proven useful to assess some features of ASM function in vivo. Despite these technologic advances, it is still not clear whether excessive narrowing in asthma is driven by ASM abnormalities, by other alterations in non-muscle factors or simply because of the overexpression of spasmogens. This is because a multitude of forces are acting on the airway wall, and because not only are these forces constantly changing but they are also intricately interconnected. To counteract these limitations, investigators have utilized in vitro and ex vivo systems to assess and compare asthmatic and non-asthmatic ASM contractility. This review describes: 1- some muscle and non-muscle factors that are altered in asthma that may lead to airway narrowing and asthma symptoms; 2- some technologies such as the forced oscillation technique that have the potential to unveil the role of ASM in airway narrowing in vivo; and 3- some data from ex vivo and in vitro methods that probe the possibility that airway hyperresponsiveness is due to the altered environment surrounding the ASM or, alternatively, to a hypercontractile ASM phenotype that can be either innate or acquired.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

Airways dilate to simulated inspiratory but not expiratory manoeuvres.

In a healthy human, deep inspirations produce bronchodilation of contracted airways, which probably occurs due to the transient distension of the airway smooth muscle (ASM). We hypothesised that deep expiratory manoeuvres also produce bronchodilation due to transient airway wall and ASM compression. We used porcine bronchial segments to assess the effects of deep inspirations, and maximal and partial expiration (submaximal) on airway calibre. Respiratory manoeuvres were simulated by varying transmural pressure using a hydrostatic pressure column: deep inspiration 5 to 30 cmH(2)O, maximal expiration 30 to -15 cmH(2)O, partial expiration 10 to -15 cmH(2)O; amidst a background of tidal oscillations, 5 to 10 cmH(2)O at 0.25 Hz. Changes in luminal cross-sectional area in carbachol-contracted airways were measured using video endoscopy. Deep inspirations produce an immediate bronchodilation (∼40-60%, p=0.0076) that lasts for up to 1 min (p=0.0479). In comparison, after maximal expiration there was no immediate change in airway calibre; however, a delayed bronchodilatory response was observed from 4 s after the manoeuvre (p=0.0059) and persisted for up to 3 min (p=0.0182). Partial expiration had little or no effect or airway calibre. The results observed demonstrate that the airway wall dilates to deep inspiration manoeuvres but is unresponsive to deep expiratory manoeuvres.

Distribution of airway narrowing responses across generations and at branching points, assessed in vitro by anatomical optical coherence tomography.

BACKGROUND: Previous histological and imaging studies have shown the presence of variability in the degree of bronchoconstriction of airways sampled at different locations in the lung (i.e., heterogeneity). Heterogeneity can occur at different airway generations and at branching points in the bronchial tree. Whilst heterogeneity has been detected by previous experimental approaches, its spatial relationship either within or between airways is unknown. METHODS: In this study, distribution of airway narrowing responses across a portion of the porcine bronchial tree was determined in vitro. The portion comprised contiguous airways spanning bronchial generations (#3-11), including the associated side branches. We used a recent optical imaging technique, anatomical optical coherence tomography, to image the bronchial tree in three dimensions. Bronchoconstriction was produced by carbachol administered to either the adventitial or luminal surface of the airway. Luminal cross sectional area was measured before and at different time points after constriction to carbachol and airway narrowing calculated from the percent decrease in luminal cross sectional area. RESULTS: When administered to the adventitial surface, the degree of airway narrowing was progressively increased from proximal to distal generations (r = 0.80 to 0.98, P < 0.05 to 0.001). This 'serial heterogeneity' was also apparent when carbachol was administered via the lumen, though it was less pronounced. In contrast, airway narrowing was not different at side branches, and was uniform both in the parent and daughter airways. CONCLUSIONS: Our findings demonstrate that the bronchial tree expresses intrinsic serial heterogeneity, such that narrowing increases from proximal to distal airways, a relationship that is influenced by the route of drug administration but not by structural variations accompanying branching sites.

Airway narrowing assessed by anatomical optical coherence tomography in vitro: dynamic airway wall morphology and function.

Regulation of airway caliber by lung volume or bronchoconstrictor stimulation is dependent on physiological, structural, and mechanical events within the airway wall, including airway smooth muscle (ASM) contraction, deformation of the mucosa and cartilage, and tensioning of elastic matrices linking wall components. Despite close association between events in the airway wall and the resulting airway caliber, these have typically been studied separately: the former primarily using histological approaches, the latter with a range of imaging modalities. We describe a new optical technique, anatomical optical coherence tomography (aOCT), which allows changes at the luminal surface (airway caliber) to be temporally related to corresponding dynamic movements within the airway wall. A fiber-optic aOCT probe was inserted into the lumen of isolated, liquid-filled porcine airways. It was used to image the response to ASM contraction induced by neural stimulation and to airway inflation and deflation. Comparisons with histology indicated that aOCT provided high-resolution images of the airway lumen including mucosal folds, the entire inner wall (mucosa and ASM), and partially the cartilaginous outer wall. Airway responses assessed by aOCT revealed several phenomena in "live" airways (i.e., not fixed) previously identified by histological investigations of fixed tissue, including a geometric relationship between ASM shortening and luminal narrowing, and sliding and bending of cartilage plates. It also provided direct evidence for distensibility of the epithelial membrane and anisotropic behavior of the airway wall. Findings suggest that aOCT can be used to relate changes in airway caliber to dynamic events in the wall of airways.

Effects of simulated tidal and deep breathing on immature airway contraction to acetylcholine and nerve stimulation.

BACKGROUND AND OBJECTIVE: In adults, respiratory movements, such as tidal and deep breaths, reduce airway smooth muscle force and cause bronchodilation. Evidence suggests that these beneficial effects of oscillatory strain do not occur in children, possibly because of reduced coupling of the airways to lung tissue or maturational differences in the intrinsic response of the airways to oscillatory strain. METHODS: The bronchodilator effects of oscillatory strain were compared in isolated airway segments from immature (3-4 weeks and 8-10 weeks old) and mature (18-20 weeks old) pigs. The lumen of fluid-filled bronchi was volume-oscillated to simulate tidal breaths and 0.5x, 2x and 4x tidal volumes. Contractions to acetylcholine and electrical field stimulation were recorded from the lumen pressure and were compared under oscillating and static conditions. Airway stiffness was determined from the amplitude of the lumen pressure cycles and the volume of oscillation. RESULTS: Volume oscillation reduced contractions to acetylcholine and electrical field stimulation in an amplitude-dependent manner and the percentage reduction was the same for the different stimuli across all age groups. There was no difference in the relaxed dynamic stiffness of airways from the different age groups. CONCLUSIONS: The intrinsic response of the airway wall to equivalent dynamic strain did not differ in airways from pigs of different ages. These findings suggest that mechanisms external to the airway wall may produce age-related differences in the response to lung inflation during development.

Mechanisms of heme iron absorption: current questions and controversies.

Iron is a critical micronutrient, and iron derived from heme contributes a large proportion of the total iron absorbed in a typical Western diet. Heme iron is absorbed by different mechanisms than non-heme iron, but despite considerable study over many years these mechanisms remain poorly understood. This review provides an overview of the importance of heme iron in the diet and discusses the two prevailing hypotheses of heme absorption; namely receptor mediated endocytosis of heme, and direct transport into the intestinal enterocyte by recently discovered heme transporters. A specific emphasis is placed on the questions surrounding the site of heme catabolism and the identity of the enzyme that performs this task. Additionally, we present the hypothesis that a non-heme iron transport protein may be required for heme iron absorption and discuss the experiences of our laboratory in examining this hypothesis.

Maintenance of airway caliber in isolated airways by deep inspiration and tidal strains.

Deep inspirations (DIs) are large periodic breathing maneuvers that regulate airway caliber and prevent airway obstruction in vivo. This study characterized the intrinsic response of the intact airway to DI, isolated from parenchymal attachments and other in vivo interactions. Porcine isolated bronchial segments were constricted with carbachol and subjected to transmural pressures of 5-10 cmH2O at 0.25 Hz (tidal breathing) interspersed with single DIs of amplitude 5-20 cmH2O, 5-30 cmH2O, or 5-40 cmH2O (6-s duration) or DI of amplitude 5-30 cmH2O (30-s duration). Tidal breathing was ceased after DI in a subset of airways and in control airways in which no DI was performed. Luminal cross-sectional area was measured using a fiber-optic endoscope. Bronchodilation by DI was amplitude dependent; 5-20 cmH2O DIs produced less dilation than 5-30 cmH2O and 5-40 cmH2O DIs (P=0.003 and 0.012, respectively). Effects of DI duration were not significant (P=0.182). Renarrowing after DI followed a monoexponential decay function to pre-DI airway caliber with time constants between 27.4+/-4.3 and 36.3+/-6.9 s. However, when tidal breathing was ceased after DI, further bronchoconstriction occurred within 30s. This response was identical in both the presence and absence of DI (P=0.919). We conclude that the normal bronchodilatory response to DI occurs as a result of the direct mechanical effects of DI on activated ASM in the airway wall. Further bronchoconstriction occurs by altering the airway wall stress following DI, demonstrating the importance of continual transient strains in maintaining airway caliber.

Subcellular location of heme oxygenase 1 and 2 and divalent metal transporter 1 in relation to endocytotic markers during heme iron absorption.

BACKGROUND AND AIM: Heme is an important dietary micronutrient, although its absorptive mechanisms are poorly understood. One hypothesis suggests enterocytes take up heme by receptor-mediated endocytosis (RME) which then undergoes catabolism by heme oxygenase (HO) inside internalized vesicles. This would require the translocation of HO-1 or HO-2 to endosomes and/or lysosomes and the presence of a transporter, possibly divalent metal transporter 1 (DMT1), to transfer released iron to the cytoplasm. Currently, the location of HO-1 and HO-2 in enterocytes is unknown. METHODS: We studied the subcellular location of HO-1, HO-2, and DMT1 in the proximal small intestine of rats by confocal immunofluorescence microscopy up to 4 h after a dose of heme or ferrous iron. Double-labeling was performed with endocytotic (EEA1, Lamp1) and structural markers (F-actin). RESULTS: HO-1 was distributed evenly throughout the cytoplasm of enterocytes and did not colocalize with endocytotic markers in any condition. HO-2 staining remained constant with dosing, presenting as a dense band in the apical cytoplasm that colocalized extensively with endosomes. DMT1 staining was markedly reduced by ferrous iron, but not heme and did not exhibit colocalization with endocytotic markers. CONCLUSION: The subcellular location of HO-2 is consistent with the RME hypothesis for heme uptake and may suggest a possible role for this enzyme in heme degradation. The lack of translocation of DMT1 with heme dosing suggests another protein may be present to transport iron released from heme.

Heme in intestinal epithelial cell turnover, differentiation, detoxification, inflammation, carcinogenesis, absorption and motility.

The gastrointestinal tract is lined by a simple epithelium that undergoes constant renewal involving cell division, differentiation and cell death. In addition, the epithelial lining separates the hostile processes of digestion and absorption that occur in the intestinal lumen from the aseptic environment of the internal milieu by defensive mechanisms that protect the epithelium from being breached. Central to these defensive processes is the synthesis of heme and its catabolism by heme oxygenase (HO). Dietary heme is also an important source of iron for the body which is taken up intact by the enterocyte. This review describes the recent literature on the diverse properties of heme/HO in the intestine tract. The roles of heme/HO in the regulation of the cell cycle/apoptosis, detoxification of xenobiotics, oxidative stress, inflammation, development of colon cancer, heme-iron absorption and intestinal motility are specifically examined.

Decreased sucrase and lactase activity in iron deficiency is accompanied by reduced gene expression and upregulation of the transcriptional repressor PDX-1.

Disaccharidases are important digestive enzymes whose activities can be reduced by iron deficiency. We hypothesise that this is due to reduced gene expression, either by impairment to enterocyte differentiation or by iron-sensitive mechanisms that regulate mRNA levels in enterocytes. Iron-deficient Wistar rats were generated by dietary means. The enzyme activities and kinetics of sucrase and lactase were tested as well as the activity of intestinal alkaline phosphatase (IAP)-II because it is unrelated to carbohydrate digestion. mRNA levels of beta-actin, sucrase, lactase, and the associated transcription factors pancreatic duodenal homeobox (PDX)-1, caudal-related homeobox (CDX)-2, GATA-binding protein (GATA)-4, and hepatocyte nuclear factor (HNF)-1 were measured by real-time PCR. Spatial patterns of protein and gene expression were assessed by immunofluorescence and in situ hybridization, respectively. It was found that iron-deficient rats had significantly lower sucrase (19.5% lower) and lactase (56.8% lower) but not IAP-II activity than control rats. Kinetic properties of both enzymes remained unchanged from controls, suggesting a decrease in the quantity of enzyme present. Sucrase and lactase mRNA levels were reduced by 44.5% and 67.9%, respectively, by iron deficiency, suggesting that enzyme activity is controlled primarily by gene expression. Iron deficiency did not affect the pattern of protein and gene expression along the crypt to villus axis. Expression of PDX-1, a repressor of sucrase and lactase promoters, was 4.5-fold higher in iron deficiency, whereas CDX-2, GATA-4, and HNF-1 levels were not significantly different. These data suggest that decreases in sucrase and lactase activities result from a reduction in gene expression, following from increased levels of the transcriptional repressor PDX-1.