heparitin-sulfate and Protein-Losing-Enteropathies

Topics: Animals; Disease Models, Animal; Heparitin Sulfate; Humans; Intercellular Junctions; Interferon-gamma; Intestinal Mucosa; Mice; Protein-Losing Enteropathies; Syndecan-1; Tumor Necrosis Factor-alpha

Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-gamma, TNF-alpha, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate- or syndecan-1-deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-gamma, TNF-alpha, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients.

Topics: Animals; Anticoagulants; Cell Line; Heparin; Heparitin Sulfate; Humans; Hypertension; Interferon-gamma; Intestinal Mucosa; Mice; Mice, Knockout; Protein-Losing Enteropathies; Syndecan-1; Tumor Necrosis Factor-alpha

Protein-losing enteropathy (PLE), the loss of plasma proteins through the intestine, is a symptom in ostensibly unrelated diseases. Emerging commonalities indicate that genetic insufficiencies predispose for PLE and environmental insults, e.g. viral infections and inflammation, trigger PLE onset. The specific loss of heparan sulfate (HS) from the basolateral surface of intestinal epithelial cells only during episodes of PLE suggests a possible mechanistic link. In the first tissue culture model of PLE using a monolayer of intestinal epithelial HT29 cells, we proved that HS loss directly causes protein leakage and amplifies the effects of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha). Here, we extend our in vitro model to assess the individual and combined effects of HS loss, interferon gamma (IFNgamma), TNFalpha, and increased pressure, and find that HS plays a central role in the patho-mechanisms underlying PLE. Increased pressure, mimicking venous hypertension seen in post-Fontan PLE patients, substantially increased protein leakage, but HS loss, IFNgamma, or TNFalpha alone had only minor effects. However, IFNgamma up-regulated TNFR1 expression and amplified TNFalpha-induced protein leakage. IFNgamma and TNFalpha compromised the integrity of the HT29 monolayer and made it more susceptible to increased pressure. HS loss itself compromises the integrity of the monolayer, amplifying the effects of pressure, but also amplifies the effects of both cytokines. In the absence of HS a combination of increased pressure, IFNgamma, and TNFalpha caused maximum protein leakage. Soluble heparin fully compensated for HS loss, providing a reasonable explanation for patient favorable response to heparin therapy.

Topics: Albumins; Cell Line; Cytokines; Dose-Response Relationship, Drug; Epithelial Cells; Flow Cytometry; Genetic Predisposition to Disease; Heparin; Heparitin Sulfate; Humans; Hydrogen Peroxide; Inflammation; Interferon-gamma; Intestinal Mucosa; Microscopy, Confocal; Models, Statistical; Pressure; Protein Binding; Protein Conformation; Protein-Losing Enteropathies; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation

Protein-losing enteropathy (PLE), the excessive loss of plasma proteins through the intestine, often correlates with the episodic loss of heparan sulfate (HS) proteoglycans (HSPG) from the basolateral surface of intestinal epithelial cells. PLE onset is often associated with a proinflammatory state. We investigated whether loss of HS or treatment with the proinflammatory cytokine TNF-alpha directly causes protein leakage and whether a combination of both exacerbates this process. We established the first in vitro model of PLE and measured the flux of albumin/FITC through a monolayer of intestinal HT29 or Caco-2 cells grown on transwells and determined the integrity by transepithelial electrical resistance (TER). Loss of HS from the basolateral surface, either by heparanase digestion or by inhibition of HS synthesis, increased albumin flux 1.58 +/- 0.09-fold and reduced TER by 23.4 +/- 6.5%. TNF-alpha treatment increased albumin flux 4.04 +/- 0.03-fold and reduced TER by 75.7 +/- 4.7% but only slightly decreased HS content. The combined effects of HS loss and TNF-alpha treatment were not only additive, but synergistic, with a 7.00 +/- 0.11-fold increase in albumin flux and a 83.9 +/- 8.1% reduction of TER. Coincubation of TNF-alpha with soluble HS or heparin abolished these synergistic effects. Loss of basolateral HS directly causes protein leakage and amplifies the effects of the proinflammatory cytokine TNF-alpha. Our findings imply that loss of HSPGs renders patients more susceptible to PLE and offer a potential explanation for the favorable response some PLE patients have to heparin therapy.

Topics: Albumins; Cell Line; Glucuronidase; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Protein-Losing Enteropathies; Tumor Necrosis Factor-alpha

Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided. Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an alpha1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide. The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported. The mutations were tested for their ability to rescue faulty N:-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain. Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective. Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast. Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress. Incomplete N:-linked glycosylation of an HS core protein and/or other biosynthetic enzymes may explain the selective localized loss of HS and PLE.

Topics: Biopsy; Carboxypeptidases; Enterocytes; Fibroblasts; Glucosyltransferases; Glycosylation; Heparitin Sulfate; Histocytochemistry; Humans; Immunohistochemistry; Infant; Intestinal Mucosa; Male; Membrane Proteins; Protein-Losing Enteropathies; Saccharomyces cerevisiae

The molecular basis of protein-losing enteropathy is unknown. However it has been shown that sulphated glycosaminoglycans may be important in regulating vascular and renal albumin loss.. We describe three baby boys who presented within the first weeks of life with massive enteric protein loss, secretory diarrhoea, and intolerance of enteral feeds. All required total parenteral nutrition and repeated albumin infusions. No cause could be found in any case despite extensive investigations, including small intestinal biopsy sampling, which were repeatedly normal.. By specific histochemistry, we detected gross abnormality in the distribution of small intestinal glycosaminoglycans in all three infants, with complete absence of enterocyte heparan sulphate. The distribution of vascular and lamina propria glycosaminoglycans was, however, normal.. The presentation of these infants suggests that enterocyte heparan sulphate is important in normal small intestinal function.

Topics: Albumins; Diarrhea, Infantile; Heparitin Sulfate; Humans; Infant Nutrition Disorders; Infant, Newborn; Intestine, Small; Male; Protein-Losing Enteropathies