Evaluation of Collagen-derived Antiangiogenic factors and Matrix Metalloproteinases in Lens epithelial cells of Pediatric eyes with Persistent Fetal Vasculature (PFV) Shail A. Vasavada, DO Devarshi Gajjar, PhD Kaid Johar SR, PhD Abhay Vasavada, MS, FRCS (England) Iladevi Cataract and IOL Research Centre, Ahmedabad, India The authors have no financial interest in this presentation
Embryonic Stage : Lens covered by tunica vasculosa lentis Failure in regression of hyaloid vasculature results in a pathological condition called Persistant Fetal Vasculature (PFV) However the exact mechanism of angiogenesis and regression is not completely known Regulation of Angiogenesis Collagen Derived AAFs • Is by Proteolytic cleavage of collagen in the lens capsule • Stimulated by release of growth factors from ECM • Inhibited by release of Antiangiogenic factors (AAF) from ECM • Matrix Metalloproteases(MMPs) regulate the proteolytic cleavage of ECM • Arresten:α1 chain COLIV • Canstatin:α2 chain COLIV • Tumstatin: α3 chain COLIV • Endostatin: α1 chain COLXVIII • 1Sottile J. BiochemBiophysActa. 20044;1654(1):13-22. • 2Mundel TM, Kalluri R. Microvasc Res. 2007;74(2-3):85-9.
HypothesisAlteration in antiangiogeneic factors could cause the failure of regression of hyaloid artery in PFV patients Purpose To measure mRNA levels of Antiangiogenic factors (arresten, canstatin, tumstatin, endostatin) and Matrix Metalloproteinases (MMP-2 and MMP-9) in lens epithelial cells(LECs) of eyes with and without PFV Study Design Case-Control Study Cases – Pediatric eyes with anterior PFV (n=6) Controls – Age-matched Congenital cataracts (n=6)
Material and Methods Representative PFV Cases: membranous cataract, traction on ciliary processes, fibrovascular plaque • Anterior capsule* with LECs obtained intraoperatively from eyes with (n=6) and without PFV (n=6) • Samples processed for RNA extraction and cDNA construction * All the anterior capsules collected from pediatric eyes during the surgery were in accordance with Declaration of Helsinki. Ethical approval for the study and use of human LECs was obtained from the Institutional Ethics Committee
Quantitative real time RT-PCR performed to determine the mRNA levels of arresten, canstatin, tumstatin, endostatin, MMP-2, MMP-9, GAPDH and Beta actin (Housekeeping genes) Standardization for qRT-PCR Amplification curves Amplification curves mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between cases and controls Melting curves Melting peaks Non-parametric Mann-Whitney U test performed
Results Relative mRNA expression of AAFs and MMPs in PFV cases and Controls In eyes with PFV... Arresten 1.6 times Tumstatin 2.5 times Canstatin 2.3 times * # * *P=0.01, # P=0.02, AAFs • The mRNA level of MMP-2 was 4 times higher in PFV cases • No change was observed in MMP-9 mRNA levels (p = 0.82)
Literature Review • Speculation • In the present study we speculate that increased level of canstatin and tumstatin could be to cope up with increased angiogenesis in the pathological condition of PFV • Decreased levels of Arresten could be because of the developmental shift in collagen IV of the lens capsule 3Zhu M et al., Exp Eye Res. 2000;70(6):767-76 4Duh EJ et al., Ophthalmology. 2004; 111:1885-8 5Saint-Geniez M and D’Amore PA Int J Dev Biol 2004; 48:1045–1058 6Hackett SF et al., J CellPhysiol. 2002;192:182–187. 7Reichel MB et al., Cell Death Differ. 1998;5:156 –162. 8Thornton JD et al., Invest Ophthalmol Vis Sci. 2007 ;48:491-9 9Rutland CS et al., Mol Vis.2007;19:47-56. 10Gehlbach P et al., Neuroscience. 2006;137(2):447-61. Apoptotic and necrotic cell death of endothelial cells and pericytes of the blood vessels play an important role in the regression of hyaloid vasculature3 Potential role for endostatin in fetal vascular remodeling of the eye was shown in a patient with Knobloch syndrome and PFV4 VEGF, FGF, PEGF, Factors implicated in developmental and pathological changes in vessel formation and regression during development5 Mouse models for PFV have implicated the role of proteins, Angiopoietin-2(Ang-2)6, P537,Arf8, VEGF-A1889, NUC110
Conclusion First study on human PHPV eyes -The levels of Anti-Angiogenic Factors are altered in eyes with PHPV -AAFs appear to play a pivotal role in maintaining the lens avascularity, which gets altered during the pathogenesis of PFV Future Direction Further studies on the protein levels, growth factors and other inhibitors