title: Transforming growth factor-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans.
abstract: Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2 (rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2 (20-40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk.
authors.affiliation: Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; Department of Pediatrics, Morsani College of Medicine, University of South Florida Health, Tampa, Florida;
authors.forename: Kopperuncholan
authors.initials: K
authors.lastname: Namachivayam
authors.affiliation: Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois;
authors.forename: Hayley P
authors.initials: HP
authors.lastname: Coffing
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University School of Pharmacy, Richmond, Virginia; Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University School of Pharmacy, Richmond, Virginia;
authors.forename: Nehru Viji
authors.initials: NV
authors.lastname: Sankaranarayanan
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University School of Pharmacy, Richmond, Virginia; Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University School of Pharmacy, Richmond, Virginia;
authors.forename: Yingzi
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; Department of Pediatrics, Morsani College of Medicine, University of South Florida Health, Tampa, Florida;
authors.forename: Krishnan
authors.initials: K
authors.lastname: MohanKumar
authors.affiliation: Department of Pediatrics, NorthShore University Health System, Evanston, Illinois;
authors.forename: Brandy L
authors.initials: BL
authors.lastname: Frost
authors.affiliation: Department of Pediatrics, University of Texas Health Science Center at San Antonio, San Antonio, Texas;
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Blanco
authors.affiliation: Department of Pediatrics, Rush University Medical Center, Chicago, Illinois; and.
authors.forename: Aloka L
authors.initials: AL
authors.lastname: Patel
authors.affiliation: Department of Pediatrics, Rush University Medical Center, Chicago, Illinois; and Department of Women Children and Family Nursing, Rush University Medical Center, Chicago, Illinois.
authors.forename: Paula P
authors.initials: PP
authors.lastname: Meier
authors.affiliation: Department of Pathology, University of Illinois at Chicago, Chicago, Illinois;
authors.forename: Steven A
authors.initials: SA
authors.lastname: Garzon
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University School of Pharmacy, Richmond, Virginia; Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University School of Pharmacy, Richmond, Virginia;
authors.forename: Umesh R
authors.initials: UR
authors.lastname: Desai
authors.affiliation: Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; Department of Pediatrics, Morsani College of Medicine, University of South Florida Health, Tampa, Florida; Department of Molecular Medicine, Morsani College of Medicine, University of South Florida Health, Tampa, Florida; akhilm@health.usf.edu.
authors.forename: Akhil
authors.initials: A
authors.lastname: Maheshwari
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism; D001682:Biological Availability; D002460:Cell Line; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism*; D020345:Enterocolitis, Necrotizing / Q000378:metabolism / Q000517:prevention & control; D006801:Humans; D007231:Infant, Newborn; D007234:Infant, Premature; D007249:Inflammation / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D008262:Macrophage Activation / Q000502:physiology; D051379:Mice; D008895:Milk, Human / Q000201:enzymology / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000378:metabolism*
chemical_list: D000893:Anti-Inflammatory Agents; D011508:Chondroitin Sulfate Proteoglycans; D016328:NF-kappa B; D011994:Recombinant Proteins; D053781:Transforming Growth Factor beta2; D002810:Chondroitinases and Chondroitin Lyases
keywords: TGF-β2; breast milk; chondroitinase; inflammation; necrotizing enterocolitis