Encapsulated VEGF121-PLA microparticles promote angiogenesis in human endometrium stromal cells

Abstract

Background: In this study, Vascular Endothelial Growth Factor 121 expressed abundantly in endometrial stromal cells is encapsulated with poly-l-lactide and characterized the properties for endometrial angiogenesis. We studied the migration, proliferation and the protein levels of human immortalized endometrium stromal cells after treating the cells with recombinant Vascular Endothelial Growth Factor (200 and 500 nanogram), and poly-l-lactide loaded Vascular Endothelial Growth Factor 121 (day 1, 20 and 30). The present study explains endometrium angiogenesis because endometrium plays an important role in pregnancy.

Results: Migration and proliferation studies in endometrium cells proved the efficiency of Vascular Endothelial Growth Factor and poly-l-lactide loaded Vascular Endothelial Growth Factor 121. This proliferated and increased the migration of the cells in vitro and also activated the Protein kinase B, Phosphatidylinositol-4, 5-Bisphosphate 3-Kinase Catalytic Subunit Beta, α-Smooth muscle actin and vascular endothelial growth factor receptor 2 pathways. Western blot analysis showed the increased expression levels of kinases, smooth muscle actin and vascular endothelial growth factor receptor 2 after the treatment with Vascular Endothelial Growth Factor and poly-l-lactide loaded Vascular Endothelial Growth Factor 121 particles in comparison to the control group. The elevated levels of α-Smooth muscle actin in endometrium cells with Vascular Endothelial Growth Factor prove the regulation of angiogenesis in vitro.

Conclusion: Endometrium thickness is one of the important factors during implantation of embryo and pregnancy. Slow release of VEGF from PLA encapsulated microparticle further controls the endothelial cell proliferation and migration and helps in the promotion of angiogenesis. The combined effect studied in vitro could be used as a pro-angiogenic drug on further in vivo confirmation.

Keywords: Angiogenesis; Endometrium; Human endometrium stromal cells; Migration; Proliferation; VEGF.

Fig. 1

Diagrammatic representation of endometrium regeneration using PLA-VEGF: (1) A woman who is not able to conceive with thin endometrium condition. (2) Collection of the endometrium stromal cells (hESC) from the patient’s endometrium tissues via. hysterectomy. (3) Production of recombinant VEGF₁₂₁. (4) Encapsulation of rVEGF₁₂₁ with poly-l-lactide. (5) Injection of the PLA-VEGF₁₂₁ into the thin endometrium (uterine lining) with the help of IUI catheter. (6) After successful regeneration of the endometrium, embryo which was prepared; transferred in the uterine. (7) After the successful transfer of the healthy embryo, the woman gets pregnant. Successful implantation lies in a fertile environment (i.e., endometrium). In regenerating the endometrium, there are high chances of successful implantation and pregnancy

Fig. 2

a MTT cell proliferation assay on human endometrium stromal cells (HESC) on 200 and 500 ng concentration of recombinant VEGF₁₂₁. Absorbance read at 570 nm; data represented as mean ± S.D. from 3 replicates. Asterisks (***) indicate significant differences of p < 0.001 in comparison with 200 ng vs. 500 ng. b MTT cell proliferation assay on human endometrium stromal cells (HESC) on PLA encapsulated rVEGF₁₂₁. Cells incubated with different concentrations for 24 h and compared with controls. Absorbance read at 570 nm; data represented as mean ± S.D. from 3 replicates. Y-axis showing the proliferation increase; asterisks (***) indicate significant differences of p < 0.001 in comparison with PBS (negative control) vs. day 1, day 20, and day 30

Fig. 3

Effects of VEGF and VEGF-PLA on HESC migration: VEGF and VEGF-PLA stimulated HESC migration. HESC was grown to confluent and the monolayers were scratched with a pipette tip and stimulated with rVEGF₁₂₁, 200 ng and 500 ng concentrations, and 1, 20, and 30 days’ release of VEGF-PLA; the cells were grown for 12 h and 24 h respectively. A Single picture representation of each group is shown; wound closure images was measured using Image J software. Images of the gap area induced at 12 h and 24 h: (a) untreated cells at 12 h; (b) 200 ng of rVEGF₁₂₁ at 12 h; (c) 500 ng of rVEGF₁₂₁ at 12 h; (d) positive control; (e) PLA–VEGF microparticles on day 1 at 24 h; (f) PLA–VEGF microparticles on day 20 at 24 h; (g) PLA–VEGF microparticles on day 30 at 24 h; (h) negative control. B The histogram showing the percentage of cell-covered area for different concentration of rVEGF₁₂₁; asterisk indicates significant differences p < 0.001, p < 0.05. Cell covered percentage were compared with the untreated control (UT). Data represented (mean ± S.D., n = 3) at 12 h. Statistical significance shown is 200 ng and 500 ng of rVEGF₁₂₁ vs. untreated group. C The histogram shows the percentage of cell-covered area at 24 h treated with rVEGF₁₂₁-loaded microparticles (PLA-VEGF) collected on different days. Data represented as mean ± S.D., n = 3. Statistical significance (p < 0.001, p < 0.05) compared with the untreated control (UT)

Fig. 4

Effects of VEGF and PLA-VEGF on protein expression of α SMA, PIK3Cb, VEGFR2, and PKBa in HESC. a Western blotting images showing the levels of protein expression after stimulation with different concentrations of rVEGF₁₂₁ and PLA-VEGF microparticles release. b–e Expression levels of α SMA (b), PIK3Cb (c), VEGFR2 (d), and PKBa (e) in HESC; the expression levels normalized with GAPDH expression. Data represented as mean ± S.D., n = 3. Statistical significance (p < 0.05, p < 0.01, p < 0.001) compared (200 and 500 ng of rVEGF₁₂₁ with the positive control, while days 1, 20, and 30 with PLA-PBS (negative control))