Studies On The Role Of Axonal Guidance Factor Netrin-1 In Human Placental Vascular Growth

Adequate placental vascular beds are critical for normal fetal growth. Therefore, it is not surprising that placental vascular beds with reduced fetal oxygen and nutrient uptake are associated with conditions such as fetal growth restriction, fetal distress and preterm delivery. These observations suggest that the factors influencing placental vascular devel-opment and function have tremendous impact on fetal growth and development and, ulti-mately, affect neonatal growth and contribute to long-term complications. Preliminary researches have shown that the process of vascular growth is initiated with focal degrade-ation of the capillary vessel basement membrane, thereby creating a defect through which migrating endothelial cells form a sprout and proliferate to create a new lumen and prepare the vessels for further maturation. This event involves microvascular endothelial cells rather than endothelial cells of large vessels suggesting that human placental micro- vascular endothelial cells (HPMECs) may play a critical role in the growth of placental vessels.It is generally accepted that endothelial cells are functionally regulated by a wide range of promoting factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). In addition to these classic factors, a number of endogenous peptides have recently been reported to play important roles as potent regulators in vascular growth. Netrin-1 is a member of a family of secreted laminin-related proteins and was originally described as an axon guidance molecule of the spinal cord during development. Moreover, recent studies have demonstrated that netrin-1 is involved in the extra-neural processes, ranging from organ morphogenesis to vascular growth.In the present study, we performed an ex vivo rat aortic ring assay and an in vivo Matrigel plug assay to determine the effect of netrin-1 on angiogenesis. Importantly, we isolated and cultured HPMECs and investigated their viability, migration and tubular formation, with an attempt to examine the effects of netrin-1.Part 1 Effects of Netrin-1 on angiogenesis in ex vivo and vivoObjective:To investigate the effect of netrin-1 on angiogenesis in ex vivo and vivo.Methods:1. In Vitro Rat Aortic Ring Assay:The aorta was excised from male Sprague-Dawley (SD) rats (aged 6 weeks, weighing 210 g-230 g). After careful removal of fibroadipose tissues, the aortas were cut into cross-sections of 1 mm in length, placed on Matrigel-coated wells, covered with an additional Matrigel and subjected to gel for 30 min at 37℃. Varying amounts (10,50 and 100 ng/mL) of netrin-1 were added to the wells to a final volume of 200μL of human endothelial serum-free media. Embedded aortic rings were cultured at 37℃in 5% CO2. Microvessel outgrowth was photographed by using a phase contrast microscope.2. In Vivo Matrigel Plug Assay:Matrigel plug assay was employed to determine whether netrin-1 plays a role as a potent regulator in vascular growth. A group of 7-week-old male C57/BL6 mice were subcutaneously given, along the peritoneal midline,0.5 mL of Matrigel containing the indicated amounts of netrin-1 (50 ng/mL) and PBS. The injected Matrigel rapidly formed a single solid gel plug. After 7 days, the skin of the mouse was easily pulled back to expose the Matrigel plug which remained intact. The hemoglobin level was measured for quantifying blood vessel formation. A part of each plug was fixed with 4% paraformaldehyde, embedded in paraffin and immunostained with the anti-body for CD34 (1:100), a marker for endothelial cells.Results:1.10 ng/mL,50 ng/mL and 100 ng/mL netrin-1 increased microvessel sprouting from the adventitia of aortic rings and the activity of netrin-1 peaked at 50 ng/mL.2. The plug containing netrin-1 had a few vessels, but nothing in the control Matrigel plug. The hemoglobin of the plug containing netrin-1 and the control were 53.4±7.3,5.8±0.9, respectively.Conclusion Netrin-1 induces angiogenesis in ex vivo and vivo.Part 2 Primary culture and identification of human placental microvascular endothelial cellsObjective:To explore the methods of culture and identification of HPMVECs.Methods:1. Isolation and Culture of HPMECs:The placentas were collected aseptically from normal pregnant women and then immediately washed twice by chilled antiseptic solution containing 100 U/mL of penicillin,100 U/mL of streptomycin and 5μg/mL of amphotericin. They were immediately excised and finely minced into small fragments. The fragments were digested for 1 h in a shaking incubator (100 cycles/min) at 37℃in a mixture of 0.1% collagenase and 0.2% dispase. Tissues were further digested with 0.02% DNase for 20 min at 37℃with gentle agitation. Tissues were then treated with 0.25% trypsin for 10 min. This step was terminated by adding fetal calf serum, followed by sequential sieving of the tissue fragments through 90-,70-, and 40-μm meshes. The flow-through was centrifuged at 1000 g for 10 min at 4℃, and the pellets were collected. The pellets were gently re-suspended in 2 mL of DMEM and layered onto the surface of a gradient and centrifuged for 20 min at 400 g. The gradient was achieved by using mixture consisting of 9 portions of pure Percoll and one portion of 8.5% NaCl, diluted with 0.85% NaCl to yield three concentrations of Percoll (25%,30% and 35%). After centrifugation, a white band emerged. The band was collected and washed twice with DMEM to eliminate the Percoll. The collected cells were re-suspended in DMEM and centrifuged at 1000 g for 5 min at 4℃. The pellets were collected and re-suspended in the culture medium containing DMEM, FCS (20%), penicillin (100 U/mL), streptomycin (100 U/mL), amphotericin (5μg/mL), epidermal growth factor (10 ng/mL), hydrocortisone (25μg/mL) and heparin (25 U/mL). Cells were plated into a culture flask and subsequently incubated at 37℃in 5% CO2. The culture medium was changed after 24 h and every two days thereafter.2. Immunofluorescence:Cells grown on coverslips were rinsed twice in PBS, fixed in 4% paraformaldehyde for 15 min and washedwith PBS three times. After blocking with normal goatserum for 20 min at room temperature, the coverslips were incubated overnight at 4℃with the polyclonal rabbit antibody against human vWF diluted at 1:100 with PBS. Following theincubation, the coverslips were washed three times with PBS and incubated for 1 h at 37℃with a goat anti-rabbit IgG coupled to a green fluorescein diluted at 1:100. Before examination under a fluorescence microscope, the coverslips were washed three times with PBS.3. RT-PCR:According to a previous study, the relative mRNA expression levels of homeobox genes TLX1 and TLX2 were significantly increased in HPMECs as compared with the levels in human umbilical vein endothelial cells (HUVEC). The mRNA expression levels of TLX1 and TLX2 were measured by RT-PCR to identify HPMECs. Theβ-actin gene served as an internal control. Total RNA was isolated from the purified microvascular endothelial cells with the Total RNA Extraction Miniprep system. The first-strand complementary (cDNA) synthesis reaction was performed by using the RevertAid First Strand cDNA Synthesis Kit.Results:HPMECs were cultured in vitro and vWF was positive by immunofluorescence. Homeobox genes TLX1, TLX2 relative mRNA expression levels were significantly increased in HPMECs compared with HUVEC via RT-PCR (p<0.01 and p<0.05).Conclusion:Human placental microvascular endothelial cells can be isolated and cultured successfully in vitro.Part 3 Effects of netrin-1 on human placental microvascular endothelial cellsObjective:To assess the effects of netrin-1 on the viability, migration and tube formation of HPMECs.Methods:1. MTT Assay:The effect of netrin-1 on the viability of HPMECs was determined by the MTT assay. Cells were seeded into the wells of a 48-well plate at a concentration of 1×104 cells/well. After incubation with 10,50 and 100 ng/mL of netrin-1 for 24 h, the cells were incubated with 5 mg/mL MTT for 4 h. The reagent was reduced by living cells to from insoluble blue formazan products. The cells were then washed, solubilized with 10% sodium dodecyl sulfate, and quantified by measuring the absorbance (A value) at 570 nm.2. Cell Migration Assay:The cell migration assay was conducted in a transwell plate (pore size:8 mm). HPMECs were suspended in media at a density of 3×105 cells/mL, and 0.1 mL of the cell suspension and 10,50, and 100 ng/mL of netrin-1 were added in serum-free media to the upper compartment of the filter. Serum-free media was placed in the lower chamber. After incubation at 37℃for 6 h, filters were fixed with 8% glutaraldehyde and stained with trypan blue. The total number of migrated cells was calculated by counting five randomly selected fields.3. Tube Formation Assay:Growth factor-reduced Matrigel (300μL) was pipetted onto 24-well culture plates and polymerized for 30 min at 37℃. HPMECs were seeded onto the surface of the Matrigel, and 10,50 and 100 ng/mL of netrin-1 was then added. The plates were incubated for 8 h at 37℃. The morphological changes of the cells were observed under a microscope.4. Flow cytometric analysis:HPMECs were treated with the netrin-1 (10,50 and 100 ng/mL) for 48 h. After final treatment, cells were harvested, washed twice with ice-cold phosphate-buffered saline, fixed in 70% ethanol at 20℃. Cells were stained with propidium iodide and annexin V and then flow cytometrically examined.Results:1.10,50, and 100 ng/mL netrin-1 treatments of HPMECs caused a significant increase in the absorbance of the cells (0.324±0.02,0.503±0.019,0.447±0.032, p<0.05 and p<0.01 compared to the control).2.10,50, and 100 ng/mL netrin-1 promote the migration of HPMECs (15.6±0.5,20.8±0.9, 19.6±0.2 p<0.05 and p<0.01 compared to the control).3.10,50, and 100 ng/mL netrin-1 promotes the tube formation of HPMECs (13±1,14±2, 19±2, p<0.05 compared to the control).4. Flow cytometry showed that netrin-1 decreased the percentage of apoptotic cells (51.4±6.9,38.5±4.2,29.3±3.2 and 17.9±2.6 respectively).Conclusion:Cell culture experiments demonstrated that netrin-1 specifically promoted the viability, migration and tubular formation of HPMECs. Our findings revealed that netrin-1 is a positive factor that mediates placental vascular development. The information from this study might serve as a basis for future developmental therapeutics of placental vasculature-related diseases.