High glucose increases lysyl oxidase expression and activity in retinal endothelial cells: mechanism for compromised extracellular matrix barrier function.
OBJECTIVE
In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions.


METHODS
Rat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression.


RESULTS
Western blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability.


CONCLUSIONS
HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.
Chronopoulos
Argyrios
A
Departments of Medicine and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA.
Tang
Amanda
A
Beglova
Ekaterina
E
Trackman
Philip C
PC
Roy
Sayon
S
D000629:Aminopropionitrile / Q000494:pharmacology; D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology; D003930:Diabetic Retinopathy / Q000235:genetics / Q000473:pathology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology; D005109:Extracellular Matrix / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D008249:Protein-Lysine 6-Oxidase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000187:drug effects / Q000201:enzymology; D012172:Retinaldehyde / Q000378:metabolism
D000629:Aminopropionitrile; D008249:Protein-Lysine 6-Oxidase; D005947:Glucose; D012172:Retinaldehyde
J Clin Invest. 2007 Dec;117(12):3810-20
18037992
Coll Relat Res. 1984 Aug;4(4):239-51
6478768
J Biol Chem. 2004 Sep 24;279(39):40593-600
15277520
J Biol Chem. 2009 Jan 16;284(3):1385-93
19029090
Pathol Res Pract. 1994 Oct;190(9-10):910-9
7899140
Cancer Res. 2007 Jul 1;67(13):6278-85
17616686
Cancer Res. 2002 Aug 1;62(15):4478-83
12154058
Am J Physiol. 1995 Apr;268(4 Pt 1):C1067-74
7733229
J Clin Invest. 1990 Oct;86(4):1046-54
1976653
Oncogene. 2009 Sep 24;28(38):3390-400
19597471
Doc Ophthalmol. 1999;97(3-4):217-28
10896335
Diabetes Care. 1992 Dec;15(12):1835-43
1464241
Diabetologia. 2005 Nov;48(11):2428-36
16193290
Prog Nucleic Acid Res Mol Biol. 2001;70:1-32
11642359
J Foot Ankle Surg. 1997 Jul-Aug;36(4):272-8; discussion 330
9298442
Invest Ophthalmol Vis Sci. 1988 Dec;29(12):1836-46
3192373
Neurosci Lett. 2005 Mar 16;376(3):210-4
15721223
Cell Mol Life Sci. 2006 Oct;63(19-20):2304-16
16909208
Circulation. 2002 Nov 5;106(19):2503-9
12417550
J Histochem Cytochem. 1996 Dec;44(12):1469-79
8985139
Exp Clin Endocrinol Diabetes. 2008 Apr;116(4):198-202
18072010
Int J Cancer. 2009 Jul 15;125(2):318-27
19330836
Cancer Res. 2006 Nov 1;66(21):10238-41
17079439
Invest Ophthalmol Vis Sci. 1996 Feb;37(2):258-66
8603829
Diabetes. 2006 Jan;55(1):86-92
16380480
J Toxicol Environ Health. 1992 Nov;37(3):411-23
1359158
J Pathol. 2007 May;212(1):74-82
17354256
J Biol Chem. 1992 Apr 25;267(12):8666-71
1349020
Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9640-4
8415754
Trans Assoc Am Physicians. 1970;83:102-12
5505385
Haemostasis. 1983;13(1):36-41
6840627
Clin Exp Metastasis. 2009;26(1):35-49
18814043
Diabetes. 1994 Mar;43(3):441-6
8314018
J Biol Chem. 2001 Jun 29;276(26):24023-9
11292829
J Clin Invest. 1994 Jan;93(1):438-42
8282817
J Mol Histol. 2004 Nov;35(8-9):845-55
15609098
Mol Cell Biol. 2009 Aug;29(16):4467-83
19546236
J Biol Chem. 2003 Apr 18;278(16):14387-93
12473682
Biochim Biophys Acta. 1992 Mar 20;1138(3):173-83
1547278
J Cell Biochem. 2007 Aug 15;101(6):1338-54
17471532
J Biol Chem. 2003 Dec 26;278(52):52071-4
14551188
Drug News Perspect. 2008 May;21(4):218-24
18560621
Diabetes. 1996 Mar;45(3):348-53
8593941
Nature. 2006 Apr 27;440(7088):1222-6
16642001
J Pathol. 2003 Jul;200(4):537-46
12845621
J Biol Chem. 2001 Dec 28;276(52):48944-9
11684696
Anal Biochem. 2002 Jan 15;300(2):245-51
11779117
Annu Rev Physiol. 2001;63:471-94
11181964
Birth Defects Res C Embryo Today. 2006 Mar;78(1):47-68
16622848
Mol Vis. 2004 Oct 07;10:750-7
15496827
Biochem Biophys Res Commun. 2008 Feb 1;366(1):156-61
18060869
Cancer Cell. 2005 Sep;8(3):241-54
16169468
J Hepatol. 2005 Sep;43(3):499-507
16023247
Cancer Res. 2007 Feb 1;67(3):1105-12
17283144
Cancer Res. 2009 Aug 15;69(16):6685-93
19654310