Inhibition of proliferation of retinal vascular endothelial cells more effectively than choroidal vascular endothelial cell proliferation by bevacizumab

AIM： To evaluate the differential inhibitory effects of bevacizumab on cell proliferation of vascular endothelial growth factor （VEGF）-stimulated choroidal vascular endothelial cells （CVECs） and retinal vascular endothelial cells （RVECs） in vitro.METHODS： VEGF （400 ng/mL） enriched CVECs and RVECs were treated with escalating doses of bevacizumab （0.1, 0.5, 1, 1.5 and 2 mg/mL）. Cell proliferation changes were analyzed with WST-1 assay and trypan blue exclusion assay at 48, 72h and 1wk. Morphological changes were recorded with bright field microscopy.RESULTS： VEGF enriched RVECs showed significantly more decline of cell viability than CVECs after bevacizumab treatment. One week after treatment, RVEC cell proliferation decreased by 29.7%, 37.5%, 52.8%, 35.9% and 45.6% at 0.1, 0.5, 1.0, 1.5 and 2 mg/mL bevacizumab respectively compared to CVEC proliferation decrease of 4.1%, 7.7%, 2.4%, 4.1% and 17.7% （P〈0.05） by WST-1 assay. Trypan blue exclusion assay also revealed similar decrease in RVEC proliferation of 20%, 60%, 73.3%, 80% and 93.3% compared to CVEC proliferation decrease of 4%, 12%, 22.9%, 16.7% and 22.2% respectively （P〈0.05）. The maximum differential effect between the two cell types was observed at bevacizumab doses of 1.0 and 1.5 mg/mL at all time points. RVECs were 22 fold more sensitive （P〈0.01） compared to CVECs （52.8% vs 2.4%） at concentration of 1.0 mg/mL, and 8.7 fold more at 1.5 mg/mL （35.9% vs 4.1%） 1wk after treatment （P〈0.05 respectively）.CONCLUSION： VEGF-enriched RVECs are more susceptible to bevacizumab inhibition than CVECs at clinically used dosage of 1.25 mg and this differential sensitivity between two cell types should be taken into consideration in dosage selection.

Ly49 receptors activate angiogenic mouse DBA＋ uterine natural killer cells

In humans, specific patterns of killer immunoglobulin-like receptors （KIRs） expressed by uterine natural killer （uNK） cells are linked through HLA-C with pregnancy complications （infertility, recurrent spontaneous abortion, intrauterine growth restriction and preeclampsia）. To identify mechanisms underpinning the associations between NK cell activation and pregnancy success, pregnancies were studied in mice with genetic knockdown （KD） of the MHC-activated Ly49 receptor gene family. B6.Ly49KD pregnancies were compared to normal control B6.Ly49z29 and C57BL/6 （B6） pregnancies. At mid-pregnancy （gestation day （gd9.5））, overall uNK cell （TCRI^-CD122＋DBA＋DX5- （DBA＋DX5-）） and TCRIβ-CD122＋DBA-DX5＋ （DBA-DX5＋）） frequencies in pregnant uterus were similar between genotypes. Ly49KD lowered the normal frequencies of Ly49＋ uNK cells from 90.3% to 47.8% in DBA-DX5＋ and 78.8% to 6.3% in DBA＋DX5- uNK cell subtypes. B6.Ly49KD matings frequently resulted in expanded blastocysts that did not implant （subfertility）. B6.Ly49KD mice that established pregnancy had gestational lengths and litter sizes similar to controls. B6.Ly49KD neonates, however, were heavier than controls. B6.Ly49KD implantation sites lagged in early （gd6.5） decidual angiogenesis and were deficient in mid-pregnancy （gd 10.5） spiral arterial remodelling. Ultrastructural analyses revealed that B6.Ly49KD uNK cells had impaired granulogenesis, while immunocytochemistry revealed deficient vascular endothelial cell growth factor （VEGFA） production. Perforin and IFNG expression were normal in B6.Ly49KD uNK cells. Thus, in normal mouse pregnancies, Ly49 receptor signaling must promote implantation, early decidual angiogenesis and mid-pregnancy vascular remodelling. Disturbances in these functions may underlie the reported genetic associations between human pregnancy complications and the inability of specific conceptus MHCs to engage activating KIR on uNK cells.