title: Osmoprotectants suppress the production and activity of matrix metalloproteinases induced by hyperosmolarity in primary human corneal epithelial cells.
abstract: OBJECTIVE
  Hyperosmolarity has been recognized as a proinflammatory stress in the pathogenesis of dry eye disease. This study investigated the suppressive effect of osmoprotectants (L-carnitine, erythritol, and betaine) on the production and activity of matrix metalloproteinases (MMPs) in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.
  
  
  METHODS
  Primary HCECs were established from fresh donor limbal tissue explants. The cultures in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media with or without prior incubation with different concentrations of L-carnitine, erythritol, or betaine (2, 10, or 20 mM). The mRNA expression of the MMPs was determined with reverse transcription and quantitative real-time PCR (RT-qPCR). Protein production and activity were evaluated with immunofluorescent staining and gelatin zymography.
  
  
  RESULTS
  Hyperosmotic media (400, 450, or 500 mOsM) significantly stimulated mRNA expression of collagenase MMP-13, gelatinases MMP-9 and MMP-2, stromelysin MMP-3, and matrilysin MMP-7, mostly in an osmolarity-dependent fashion. The stimulated mRNA expression and protein production of these MMPs were significantly but differentially suppressed by L-carnitine, erythritol, or betaine, as evaluated with RT-qPCR and immunofluorescent staining. Interestingly, these osmoprotectants not only suppressed production but also inhibited activation of MMP-9 and MMP-2, as evaluated with gelatin zymography.
  
  
  CONCLUSIONS
  Our findings for the first time demonstrate that osmoprotectants, L-carnitine, erythritol, and betaine, suppress the gene expression, protein production, and enzymatic activity of MMPs in HCECs exposed to hyperosmotic stress. L-carnitine appears to have the broadest and strongest suppressive effect on these MMPs. These osmoprotectants may have potential effects in protecting ocular surface epithelia from MMP-mediated disorders in dry eye disease.
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX ; School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China.
authors.forename: Ruzhi
authors.initials: R
authors.lastname: Deng
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX ; Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Zhitao
authors.initials: Z
authors.lastname: Su
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX ; Tianjin Eye Hospital, Tianjin Medical University, Tianjin, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Hua
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX ; School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China.
authors.forename: Zongduan
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX.
authors.forename: De-Quan
authors.initials: DQ
authors.lastname: Li
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pflugfelder
mesh_terms: D001344:Autopsy; D001622:Betaine / Q000494:pharmacology*; D002331:Carnitine / Q000494:pharmacology*; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004896:Erythritol / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D061251:Primary Cell Culture; D020011:Protective Agents / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D020011:Protective Agents; D012333:RNA, Messenger; D001622:Betaine; D012965:Sodium Chloride; D053509:Matrix Metalloproteinase 13; D019278:Matrix Metalloproteinase 3; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D004896:Erythritol; D002331:Carnitine