BIGH3 protein and macrophages in retinal endothelial cell apoptosis.
Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.
Department of Biology, The University of Texas at San Antonio, One UTSA Circle BSB 1.03.36, San Antonio, TX, 78249, USA.
Albert A
AA
Mondragon
Brandi S
BS
Betts-Obregon
Robert J
RJ
Moritz
Kalpana
K
Parvathaneni
Mary M
MM
Navarro
Hong Seok
HS
Kim
Chi Fung
CF
Lee
Richard G
RG
LeBaron
Reto
R
Asmis
Andrew T
AT
Tsin
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008253:Macaca mulatta; D008264:Macrophages / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012160:Retina / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000099222:betaIG-H3 Protein
D016326:Extracellular Matrix Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein