October 2008
Volume 49, Issue 10
Free
Retina  |   October 2008
Remodeling Retinal Neovascularization by ALK1 Gene Transfection In Vitro
Author Affiliations
  • Bin Li
    From the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, the
    Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, Peoples Republic of China.
  • Wei Yin
    Department of Biochemistry, ZhongShan School of Medicine, the
  • Xun Hong
    Animal Center, Department of Pharmacology, and the
  • Yu Shi
    From the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, the
  • Hong-Sheng Wang
    Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Peoples Republic of China; and the
  • Shao-Fen Lin
    From the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, the
  • Shi-Bo Tang
    From the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, the
Investigative Ophthalmology & Visual Science October 2008, Vol.49, 4553-4560. doi:10.1167/iovs.07-0995
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    • Get Citation

      Bin Li, Wei Yin, Xun Hong, Yu Shi, Hong-Sheng Wang, Shao-Fen Lin, Shi-Bo Tang; Remodeling Retinal Neovascularization by ALK1 Gene Transfection In Vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(10):4553-4560. doi: 10.1167/iovs.07-0995.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To explore a novel strategy for balancing retinal neovascularization by assessing the role activin-like kinase receptor 1 (ALK1) plays in neovascularization in vascular endothelial growth factor (VEGF)–stimulated human retinal capillary endothelial cells (HRCECs).

methods. HRCECs were transfected with an ALK1 gene–encoding plasmid or a pSIREN-ALK1 RNAi vector and stimulated with VEGF. The mRNA and protein expression levels of ALK1, occludin, ANG2, and ALK5 were evaluated by real-time PCR and/or Western blot analysis. Microscopy techniques and flow cytometry were used to assess the effects of enhanced levels of ALK1 on migration and proliferation and the formation of tubelike structures of HRCECs.

results. The level of ALK1 in ALK1-transfected cells was significantly increased compared with that in control cells. ALK1-transfected cells exhibited increased expression of occludin and decreased expression of ANG2 and ALK5, compared with expression in the control cells. HRCECs transfected with pSIREN-ALK1 RNAi exhibited decreased expression of ALK1 and occludin and increased expression of ANG2 and ALK5 compared with the control cells. Transfection with ALK1 affected the migration and proliferation of VEGF-stimulated HRCECs. ALK1 also inhibited the formation of endothelial tubelike structures, but did allow the formation of entire vessel structures.

conclusions. Overexpression of ALK1 promoted remodeling of newly formed blood vessels and prevented further angiogenesis. These findings provide insight into the control of retinal neovascularization and demonstrate a novel strategy for maintaining a stable phase of vessel formation, allowing for effective retinal neovascularization without the common adverse effects seen in patients with diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.

Several diseases such as diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion may lead to retinal neovascularization, during which new retinal blood vessels form and typically exhibit hypogenicity and increased leakage. The latter is due to a deficiency in the basement membrane and the loss of blood–retinal barrier integrity. Retinal neovascularization is associated with dramatic complications, including vitreous hemorrhage, proliferative vitreoretinopathy, and retinal detachment, which may lead to visual impairment and blindness. 1 Retinal neovascularization therapies are needed to prevent these complications from occurring. 2 3 4  
Angiogenesis is a complex developmental process 5 6 that involves proliferation, sprout formation, and remodeling of blood vessels. It involves the production or activation of several growth factors, cell adhesion molecules, and transcription factors, such as vascular endothelial growth factor (VEGF) and its receptors (e.g., KDR and FIT-1), 7 8 angiogenic factors (angiopoietin-1 [ANG1] and angiopoietin-2 [ANG2]), endothelium-specific receptor tyrosine kinase (Tie2), 9 and transforming growth factor-β (TGF-β) and its receptor activin receptor-like kinase (ALK1), among others. 10  
VEGF plays a crucial role in retinal neovascularization, because it promotes vascular permeability 11 12 and induces the proliferation and migration of vascular endothelial cells as well as the formation of new blood vessels. 13 In pathologic cases, endothelial cells are activated by VEGF, which impedes remodeling during angiogenesis and results in leakage and severe complications. Circumventing the VEGF-mediated activation of endothelial cells or causing the activated endothelial cells to become inactive may be an effective strategy for completing the angiogenic remodeling process. Such a strategy would avoid vitreous hemorrhage, improve the oxygen supply to the retina, and ultimately promote proper retinal neovascularization. 
It has also been demonstrated that ALK1 plays an important role in the formation and development of new blood vessels. 14 ALK1 is the receptor of TGF-β, 15 16 interacts with ALK5, 17 18 and stimulates the differentiation and recruitment of periendothelial cells such as pericytes and vascular smooth muscle cells (SMCs). 19 The balance between activation of the ALK1 and ALK5 signaling pathways in endothelial cells is crucial for determining the vascular endothelial properties during angiogenesis. 20 In addition, ALK1 signaling plays a role in the maturation of arterial vessels, as its overexpression results in the formation of therosclerotic lesions and promotes mesenchymal cell proliferation and SMC differentiation. 21 Furthermore, ALK1 has been demonstrated to play a role in vascular remodeling. 22 Studies of ALK1 gene knockout mice have revealed that the expression of genes encoding the angiogenesis-related factors VEGF and ANG2 were upregulated and that expression patterns correlate with enhanced blood vessel permeability. 23 24 Mutations in the ALK1 gene can cause type II hereditary hemorrhagic telangiectasia. 25 Other reports have indicated that ALK1 plays a role in the resolution phase rather than in the activation phase of angiogenesis. 19 ALK1 is also involved in mediating the effect of bone morphogenic protein on endothelial cells. 26 Furthermore, it has been demonstrated that occludin, an important factor in the formation of the blood–retinal barrier, plays a key role in preventing retinal blood vessel leakage. 27  
Since retinal neovascularization is a compensatory response to the shortage of retinal blood and oxygen supply, inhibition of angiogenesis is not the most effective approach to ameliorating the oxygen shortage. In this study, we explored a novel strategy of maintaining a stable phase of retinal neovascularization and investigated the role of ALK1 in neovascularization in retinal cells that are activated by VEGF (this mimics the pathologic state). 
Methods
Culture of Human Retinal Endothelial Cells
Ten donor eyes were obtained from the Zhongshan Ophthalmic Center Eye Bank and were managed in accordance with the guidelines in the Declaration of Helsinki for research involving human tissue. The protocol was approved by the institutional review board. Donors were healthy accident victims with an average age of 36.8 years. HRCEC cultures were established as published. 28 Briefly, the donor eyes were cut circumferentially 3 mm posterior to the limbus, and the retinas were harvested and homogenized by two gentle up-and-down strokes in a 15-mL homogenizer (Dounce; Bellco Glass Co., Vineland, NJ). The homogenate was filtered through an 88-μm sieve. The remaining retentate was digested in 0.066% collagenase for 45 minutes at 37°C. The homogenate was centrifuged (1000g for 10 minutes), and the pellet was resuspended in human serum-free endothelial-basal growth medium (Invitrogen-Gibco, Grand Island, NY), supplemented with 20% fetal bovine serum, 50 U/mL endothelial cell growth factor (Sigma-Aldrich, St. Louis, MO), and 1% insulin-transferrin-selenium. Cells were cultured in fibronectin-coated dishes and incubated at 37°C in a humidified atmosphere containing 5% CO2
Characterization of HRCECs
Cultured cells were characterized for endothelial homogeneity by evaluating their activity to factor VIII antigen (von Willebrand factor) and determining unchanged morphology under culture conditions by light microscopy. The expression of acetyl-LDL (Ac-LDL) receptors in endothelial cells was measured by adding fluorescence-labeled AC-LDL (Biomedical Technologies, Palatine, IL). Only cells from passages 3 to 7 were used in the experiments. 
ALK1-Plasmid Vectors for Transfection
The full-length coding sequence of ALK1 (1511 bp) was isolated from a human cDNA library. The sequences of the primers used for cloning into pcDNA3.1 (Invitrogen, Carlsbad, CA) were sense, 5′-GAGAAT TCACCATG ACCTTGGG CTCCCC-3′, and antisense, 5′-ACTCGAGCTATTGAATCACTT TAGGC-3′. All samples were amplified by standard PCR performed in a final volume of 50 μL. The cycling protocol used for the PCR was 30 cycles of 94°C for 30 seconds, 59°C for 45 seconds, and 72°C for 1 minute. The amplified DNA was separated on a 1% agarose gel by electrophoresis and stained with ethidium bromide. The signal was quantified by densitometry (Kontron IBAS2.0; Carl Zeiss Meditec, GmbH Jena, Germany). The full-length coding sequence of ALK1 was cloned into pcDNA3.1 + vectors under the control of the cytomegalovirus promoter by using standard cloning procedures. The vector, PEGFPC2, was used to monitor the transfection efficiency by visualization of EGFP. 
Transfection of VEGF-Stimulated HRCECs
VEGF (10 ng/mL) was added to cultured HRCECs to activate the cells. VEGF-stimulated HRCECs were then transfected with pcDNA3.1+ALK1 or PEGFPC2 (to monitor transfection) by using a transfection reagent (Lipofectamine 2000; Life Technologies, Inc., Gaithersburg, MD). The cells were plated in 24-well culture plates and grown overnight to 70% to 80% confluence. They were then washed twice with human serum-free endothelial-basal growth medium, overlaid with a mixture of plasmid DNA and the transfection reagent (at a ratio of 1 μg to 2 μL) in the serum-free growth medium. The medium was changed after 4 hours. HRCECs transfected with pcDNA3.1+ALK1 were compared with HRCECS transfected with pcDNA3.1+vector alone (as the vector control). 
Real-Time PCR to Determine ALK1, ANG2, ALK5, and Occludin Expression
VEGF-stimulated HRCECs were transfected with pcDNA3.1+ALK1 or pcDNA 3.1+vector control and were cultured in six-well plates. After 24 hours, cells from six wells per group were harvested, and RNA was extracted with a kit (Qiagen, Hong Kong, China). After quantitation of the RNA, reverse transcription was performed to retrotranscribe the RNA into cDNA (Super Script II RT kit; Invitrogen). Real-time PCR (RT-PCR) was performed with a Taq polymerase kit (Hotstar; Qiagen) with SYBR Green technology (Applied Biosystems Inc., Foster City, CA). 
β-Actin was used as an internal control for the PCRs. Primers for the PCRs were as follows: β-actin sense, 5′-TGAGACCTTCAACACCCCAG-3′, and antisense, 5′-GCCATCTCTTGCTCGAAGTC-3′; ALK1 sense, 5′-GCAACCTGCAGTGTTGCATC-3′; and antisense, 5′-CGGATCTGCTCGTCCAGCAC-3′; occludin sense, 5′-TGCATGTTCGACCAATGC-3′; and antisense, 5′-AAGCCACTTCCTCCATAAGG-3′; ANG2 sense, 5′-CGACTTCAAGAGCCGCAATGT-3′; and antisense, 5′-GTCCGGCGGGCAATC TC-3′; and ALK5 sense, 5′-GCCGTTTGACTGAAGGCT G-3′, and antisense, 5′-GGGCATCCCAAGCCTCATC-3′. 
The PCR reaction for ALK1 was performed in a final reaction volume of 50 μL in the following conditions: a preheating cycle at 94°C for 3 minutes, then 30 cycles of 94°C for 1 minute, 55°C for 30 seconds, and 72°C for 45 seconds and finally elongation at 72°C for 8 minutes. The PCR conditions for occludin and ALK5 were identical, except that 28 cycles were run, and the annealing temperature was 56°C. Melting-curve analysis was performed by monitoring FAM/SYBR fluorescence. 
Western Blot Analyses
Total proteins were extracted from transfected HRCECs and quantitated using the bicinchoninic acid (BCA) method. An amount of 50 μg total protein was mixed with loading buffer, denatured for 5 minutes at 60°C, cooled, centrifuged for 5 minutes, and separated by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibodies of mouse anti-human ALK1 monoclonal antibody (1500× dilution; DaAn Gene Co., Guangzhou, China), anti-human ANG2 (1500× dilution; BD BioSciences, San Jose, CA), anti-human occludin (1000× dilution; BD BioSciences), and anti-human ALK5 monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used for probing the proteins. A secondary antibody (1000× dilution) was then applied, and the signal was revealed by chemiluminescence. The same polyvinylidene difluoride (PVDF) membrane was reused to detect β-actin by incubation with mouse anti-human actin antibody (Gene Co., Hong Kong, China) which was used as an internal control. The bands observed on the films were analyzed by automatic image analysis, and the integrated optical density (OPTDI) of each ALK1 band was normalized to the OPTDI value of the corresponding β-actin band from the same sample. 
Inhibition of ALK1 Expression with RNA Interference (RNAi)
A vector containing a small inhibitory RNA for ALK1 was constructed by cloning the double-stranded sequence: 5′-gatccGTCTCATCCTGAAAGCATCTTCAAGAGAGATGCTTTCAGGATGAGACTTTTTTACGCGTg-3′, 5′-aattcACGCGTAAAAAAGTCTCATCCTGAAAGCATCTCTCTTGAAGATGCTTTCAGGATGAGACg-3′ (Qiagen) into a vector (RNAi-Ready pSIREN-RetroQ ZsGreen pSIREN vector; BD BioSciences) to yield the pSIREN-ALK1 RNAi vector. The pSIREN-ALK1 RNAi or pSIREN vectors were transfected into HRCECs under stimulation of VEGF by using the transfection reagent, as described earlier. Thereafter, Western blot analyses were performed to examine the expression levels of ALK1, occludin, ALK5, and ANG2, as previously described. 
Cell Cycle Analysis
Cell cycle analysis was performed using propidium iodide (PI) to visualize DNA. The cells were trypsinized, pelleted, and resuspended in one volume of ice-cold phosphate-buffered saline (PBS). The suspension was gently agitated while three volumes of ice-cold 95% ethanol was slowly added. Cold reagents and the gradual addition of ethanol were used to reduce clumping of cells. The cells were then pelleted and resuspended in equal volumes of 30 μg/mL PI and 100 μg/mL RNase A, both in PBS. Stained cells were stored overnight at 4°C and protected from light until analysis. Flow cytometry was performed to determine the percentage of cells in the G1 and S phases of the cell cycle. 
EC Migration Assay
Migration was measured in a Boyden chamber assay. A filter (8-μm pore; Nucleopore; Corning Costar, Corning, NY) was coated with fibronectin. HRCECs transfected with pcDNA3.1+ALK1 or pSIREN-ALK1 RNAi and the respective vector control cells (pcDNA3.1+vector or pSIREN vector) were trypsinized and suspended at a final concentration of 1 × 106 cells/mL in 200 μL serum-free endothelial basal medium. The chambers were put in six-well plates and cultured overnight. After 24 hours, the cells were fixed in 3.7% formaldehyde and stained with hematoxylin and eosin (HE). The cells on the upper surface of the filter were removed with a cotton swab. The cells on the lower surface of the filter were quantitated by counting five random fields. 
Tubelike Structure Formation Assay
Tubelike structure (TLS) assays were performed as has been published. 29 30 Briefly, the cells were plated in 0.33 cm2 polycarbonate filter chambers (Transwell; Corning) using 0.4-μm pore filters coated with synthetic basement membrane (Matrigel; BD BioSciences). The cultures were maintained for 3 days in serum-free endothelial basal medium. Photomicrographs of the developing tube networks were sketched with a digitizing tablet by an observer blinded to the experimental conditions. The number of branch points was then counted, and the average interbranch distance calculated. Three to five random fields were quantitated for each chamber. 
Statistical Analyses
Commercial software (SPSS 15.0; SPSS Inc, Chicago, IL) was used to compare the expression of ALK1, occludin, ANG2, and ALK5 in the ALK1 transfection experiments and in the two-dimensional blood vessel formation assays, by using an independent, two-sample Student’s t-test. The number of migrating cells was compared by analysis of variance (ANOVA). Continuous variables are presented as the mean ± SD. All statistical assessments were two-sided and evaluated at the 0.05 level of significant difference. 
Results
Characterization of Established HRCECs and Transfection of VEGF-Stimulated HRCECs and Overexpression of ALK1
Established HRCECs were initially evaluated by immunolocalization, to demonstrate that LDL receptors (red fluorescence; Fig. 1A ) and factor VIII antigen (von Willebrand Factor, green fluorescence; Fig. 1B ), both frequently used as endothelial cell markers, are present in the cytoplasm of VEGF-stimulated HRCECs. HRCECs were stimulated with VEGF and subsequently transfected with pcDNA3.1+ALK1 or PEGFPC2, which caused the ALK1 gene to be overexpressed. The transfection efficiency was 42%, as determined 2 days after transfection. 
The expression level of ALK1 in VEGF-stimulated HRCECs was confirmed by RT-PCR and Western blot analyses. RT-PCR revealed that the ratio of ALK1/β-actin in the ALK1 transfection group was significantly higher than that in the control group (P = 0.022; Table 1 ). Western blot analyses confirmed that transfection with the ALK1 gene led to a significant increase in the protein levels of ALK1 in ALK1-transfected HRCECs compared with levels in the control group (P = 0.005, Fig. 2A ; Table 2 ). 
Correlations between the Levels of ALK1 and Occludin, ANG2, and ALK5
The expression of occludin in VEGF-stimulated HRCECs that overexpressed ALK1 was evaluated by real-time PCR and Western blot analyses. RT-PCR demonstrated that the occludin/β-actin ratio in VEGF-stimulated HRCECs overexpressing ALK1 significantly increased compared with that in the control group (Table 1) . This was confirmed by Western blot analyses (Fig. 2B , Table 2 ). Our results indicate that ALK1 stimulated the expression of occludin in VEGF-activated HRCECs. Furthermore, we noticed a negative correlation between the ALK1 level and that of ANG2 and ALK5. RT-PCR revealed that the ANG2/β-actin and ALK5/β-actin ratios were significantly decreased when ALK1 was overexpressed compared with expression in the control group (Table 1) , and their protein expression was confirmed by Western blot analyses (Figs. 2C 2D ; Table 2 ). 
When pSIREN-ALK1 RNAi was transfected in VEGF-stimulated HRCECs, the expression of ALK1was shown by Western blot to be significantly inhibited compared with that in the control group (with pSIREN vector; Table 3 ). The ratio of occludin/β-actin significantly decreased in the pSIREN-ALK1 RNAi group compared with that in the control group (Figs. 3A 3B ; Table 3 ). However, the ANG2/β-actin and ALK5/β-actin ratios were significantly increased in the pSIREN-ALK1 RNAi group compared with those in the control group (Figs. 3C 3D ; Table 3 ). 
ALK1 Inhibition of the Migration and Proliferation of VEGF-Stimulated HRCECs
The effects of ALK1 on VEGF-stimulated HRCEC migration was investigated in a Boyden chamber assay. Cells from the pcDNA3.1+ALK1 and pcDNA3.1 vector control groups were placed in the Boyden chambers. The migration of HRCECs was observed 24 hours later. The number of cells that migrated in the pcDNA3.1+ALK1 transfection group was significantly lower (12.50 ± 1.87) than in the control group (23.83 ± 4.31, P < 0.001, Figs. 4A 4B ). The number of cells that migrated in the ALK1 RNAi–transfected group (37.67 ± 4.68) was significantly higher than in the pSIREN vector control group (26.46 ± 2.39, P < 0.001, Figs. 4C 4D ). 
Cell proliferation was evaluated by flow cytometry. It was found that that 61.37% ± 2.25% of VEGF-stimulated HRCECs overexpressing ALK1 were in the G0/G1 cell cycle phase in contrast to only 40.21% ± 3.59% of control HRCECs. For the RNAi experiments, 28.52% ± 2.32% of pSIREN-ALK1 RNAi transfected HRCECs and 42.62% ± 3.11% of pSIREN vector–transfected HRCECs were in the G0/G1 cell-cycle phase (Table 4) . These findings suggest that ALK1 inhibits the proliferation of VEGF-stimulated HRCECs at G0/G1
ALK1 Inhibition of the Formation of VEGF-Stimulated Endothelial TLSs
Two-dimensional TLSs were generated by using an assay employing VEGF-stimulated HRCECs. The number of TLSs in the pcDNA3.1+ALK1 transfection and pcDNA3.1 vector control groups were counted after 3 days’ incubation and were found to be 7.67 ± 1.21 and 15.33 ± 2.81, respectively (P = 0.001, Figs. 5A 5B ). VEGF-stimulated HRCECs transfected with pSIREN-ALK1 RNAi exhibited enhanced growth that resulted in cell fusion that affected the formation of TLSs. The number of TLSs in the pSIREN vector transfection group was 12.54 ± 2.37 (Figs. 5C 5D) . These results suggest that ALK1 significantly inhibits the formation of TLSs in HRCECs induced by VEGF. In addition, this experiment illustrates that in the ALK1 transfection group, an entire vessel structure was generated. This result contrasts with that in the control group, in which formation of a relatively incomplete vessel structure was observed. 
Discussion
To date, most studies in which treatment strategies to ameliorate retinal neovascularization were examined focused on inhibiting angiogenesis, 4 31 similar to the strategies used for the treatment of neovascularization in tumors. 32 Neovascularization in tumors is important for nutrient supply maintenance, and it is therefore unsurprising that inhibition of neovascularization in tumors can effectively suppress tumor development. Inhibiting angiogenesis is not sufficient to ameliorate retinal neovascularization, which is a compensatory response to the shortage of blood and oxygen. Remodeling of newly formed retinal blood vessels may prevent the complications induced by blood vessel leakage. Moreover, it may improve retinal nutrition and oxygen supply, resulting in enhanced function. Others have reported that the inhibition of retinal neovascularization is indeed not the optimal strategy, 33 as it appears that imbalances between stimulatory and inhibitory proteins contribute to retinal neovascularization. Therefore, re-establishing this balance by ocular gene transfer to block stimulators or increase expression of endogenous inhibitors is a potential therapeutic approach. This was the thesis of our current research. 
ALK1 plays a key role in blood vessel remodeling and maintaining stability. ALK1 signaling promotes endothelial cell migration and proliferation by upregulating Id1, a transcription factor promoting vascular growth through repressing an inhibitor of angiogenesis, thrombospondin-1. 34 35 However, several contrasting observations have been reported. ALK1 was shown to stimulate endothelial cell proliferation and migration. 34 Other studies indicated that ALK1 suppressed endothelial cell proliferation and migration, in contrast to ALK5, which was shown to stimulate those processes. 36 37 We previously demonstrated that ALK1 could stimulate normal endothelial cells to proliferate and migrate. 38 More interesting, in the present study that ALK1 inhibited the proliferation and migration of VEGF induced HRCECs. Scharpfenecker et al. 39 reported that ALK1 can inhibit the proliferation of bFGF-induced endothelial cells and VEGF-induced angiogenesis. Therefore, based on our observations and other findings, we postulated that ALK1 may elicit different effects in endothelial cells, depending on the condition of those cells. 34 36 37 40 41 42 43 44 45 46 47 Enhanced levels of ALK1 inhibit ALK5 and stimulate the Samd1/5 pathway. The latter has no significant effect on ANG2 and VEGF (with low expression) in HRCECs under normal conditions. This, together with the fact that TGF-β and the Smad2/3 pathway inhibit the formation of endothelin-1, leads to endothelial cells that exhibit limited proliferation and migration. In contrast, the Smad1/5-mediated inhibition of ANG2 and VEGF (with high expression) in HRCECs that are activated (e.g., stimulated by VEGF) affects endothelial cell proliferation and migration (Fig. 6)
The effects of ALK1 on blood vessel remodeling were confirmed by examining the effects of ALK1 on endothelial cells under different conditions. ALK1 may stimulate the differentiation of interstitial cells into endothelial cells and repair the blood vessels when damage occurs. In the process of proliferation, ALK1 may suppress angiogenesis and maintain the stability of blood vessels by inhibiting the proliferation and migration of activated endothelial cells. Remodeling of retinal neovascularization is based on these effects of ALK1. The endothelial cells are activated in newly formed blood vessels; however, the remodeling that occurs in retinal neovascularization cannot be completed because of the continuous proliferation. ALK1 inhibited the proliferation and migration of VEGR-stimulated HRCECs and suppressed the formation of TLSs. It appears that ALK1 induces endothelial cells in newly formed blood vessels to return to the inactive state and facilitates the completion of the remodeling of these blood vessels, therefore avoiding additional angiogenesis. Our results indicated that ALK1 increased the expression of occludin in VEGF-stimulated HRCECs. Occludin is an important factor in the formation of the blood–retinal barrier and plays a key role in preventing retinal blood vessel leakage. 27 ALK1 may upregulate the expression of occludin and promote the formation of the blood–retinal barrier and, as a consequence, decrease blood vessel leakage. 
In the case of diabetic retinopathy, retinal neovascularization is a compensatory response to the shortage of blood and oxygen supply. Concomitantly, angiogenic factors such as VEGF and ANG2 are upregulated. We demonstrated that overexpression of ALK1 in VEGF-stimulated HRCECs inhibited HRCEC proliferation and migration. Moreover, ALK1 increased the expression of occludin. Occludin is an important mediator of blood–retinal barrier formation and plays a key role in preventing retinal blood vessel leakage, thus facilitating the integrity and maturation of newly formed blood vessels. The latter ameliorates complications such as hemorrhage. Like normal blood vessels, the newly formed blood vessels can mediate nutritional supply to the retina and help maintain function. 
In summary, overexpression of ALK1 promoted the remodeling of newly formed blood vessels and prevented further angiogenesis. Our findings provide insight into the control of retinal neovascularization and demonstrate a novel strategy for maintaining a stable phase of vessel formation, allowing for effective retinal neovascularization without the common adverse effects seen in patients with diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion. 
 
Figure 1.
 
In vitro identification of VEGF-stimulated HRCECs. (A) Immunolocalization of LDL receptors represented by red fluorescent signal in the cytoplasm. (B) Immunolocalization of factor VIII antigen (von Willebrand Factor) represented by green fluorescent signal in the cytoplasm. The blue fluorescent regions indicate nuclei that were stained with Hoechst 33258. Magnification, ×200.
Figure 1.
 
In vitro identification of VEGF-stimulated HRCECs. (A) Immunolocalization of LDL receptors represented by red fluorescent signal in the cytoplasm. (B) Immunolocalization of factor VIII antigen (von Willebrand Factor) represented by green fluorescent signal in the cytoplasm. The blue fluorescent regions indicate nuclei that were stained with Hoechst 33258. Magnification, ×200.
Table 1.
 
Evaluation by Real-Time PCR of ALK1, Occludin, ANG2, and ALK5 Expression in VEGF-Stimulated HRCECs That Overexpress ALK1
Table 1.
 
Evaluation by Real-Time PCR of ALK1, Occludin, ANG2, and ALK5 Expression in VEGF-Stimulated HRCECs That Overexpress ALK1
Gene Control HRCECs (n = 6) ALK1 Transfected HRCECs (n = 6) P
ALK1/β-actin 0.81 ± 0.09 1.05 ± 0.18 0.022
Occludin/β-actin 0.84 ± 0.07 1.23 ± 0.06 <0.001
ANG2/β-actin 0.74 ± 0.09 0.47 ± 0.05 <0.001
ALK5/β-actin 0.70 ± 0.03 0.42 ± 0.05 <0.001
Figure 2.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in pcDNA3.1+ALK1 transfected HRCECs and control cells under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 2 ).
Figure 2.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in pcDNA3.1+ALK1 transfected HRCECs and control cells under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 2 ).
Table 2.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2 and ALK5 Levels in VEGF-Stimulated HRCECs Producing a Higher Level of ALK1
Table 2.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2 and ALK5 Levels in VEGF-Stimulated HRCECs Producing a Higher Level of ALK1
Gene Control HRCECs (n = 6)* ALK1-Transfected HRCECs (n = 6) P
ALK1/β-actin 0.74 ± 0.08 0.90 ± 0.07 0.005
Occludin/β-actin 0.45 ± 0.08 0.77 ± 0.10 0.001
ANG2/β-actin 0.33 ± 0.03 0.16 ± 0.03 0.001
ALK5/β-actin 0.74 ± 0.07 0.43 ± 0.06 0.001
Table 3.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2, and ALK5 Levels in VEGF-Stimulated ALK RNAi Transfected HRCECs
Table 3.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2, and ALK5 Levels in VEGF-Stimulated ALK RNAi Transfected HRCECs
Gene Control HRCECs with pSIREN Vector (n = 6)* ALK1 RNAi Transfected HRCECs (n = 6) P
ALK1/β-actin 0.73 ± 0.07 0.31 ± 0.06 0.001
Occludin/β-actin 0.93 ± 0.09 0.35 ± 0.07 0.001
ANG2/β-actin 0.87 ± 0.13 1.09 ± 0.09 0.007
ALK5/β-actin 0.83 ± 0.08 1.17 ± 0.11 0.001
Figure 3.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in ALK1 RNAi–transfected HRCECs and pSIREN vector under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 3 ).
Figure 3.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in ALK1 RNAi–transfected HRCECs and pSIREN vector under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 3 ).
Figure 4.
 
Evaluation of the effect of ALK1 on the migration of VEGF-stimulated HRCECs. HRCECs were transfected with pcDNA3.1+ALK1, pSIREN-ALK1 RNAi, or vector control, and the cell migration was assessed with the Boyden chamber assay. Cells were fixed in 3.7% formaldehyde and stained with HE. Migration of HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector, (C) pSIREN-ALK1 RNAi, or (D) pSIREN vector. The number of migrating cells in the groups was 12.50 ± 1.87, 23.83 ± 4.31, 37.67 ± 4.68, and 26.46 ± 2.39 (P < 0.001), respectively.
Figure 4.
 
Evaluation of the effect of ALK1 on the migration of VEGF-stimulated HRCECs. HRCECs were transfected with pcDNA3.1+ALK1, pSIREN-ALK1 RNAi, or vector control, and the cell migration was assessed with the Boyden chamber assay. Cells were fixed in 3.7% formaldehyde and stained with HE. Migration of HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector, (C) pSIREN-ALK1 RNAi, or (D) pSIREN vector. The number of migrating cells in the groups was 12.50 ± 1.87, 23.83 ± 4.31, 37.67 ± 4.68, and 26.46 ± 2.39 (P < 0.001), respectively.
Table 4.
 
Effect of ALK1 on the Proliferation of VEGF-Stimulated HRCECS
Table 4.
 
Effect of ALK1 on the Proliferation of VEGF-Stimulated HRCECS
Cell Cycle Phase (%) ALK1-Transfected HRCECs (n = 6) Control HRCECs (n = 6) ALK1 RNAi HRCECs (n = 6) pSIREN Vector HRCECs (n = 6)
G0/G1 61.37 ± 2.25 40.21 ± 3.59 28.52 ± 2.32 42.62 ± 3.11
S 15.46 ± 2.08 25.42 ± 3.84 36.18 ± 3.60 23.05 ± 3.16
G2+M 23.17 ± 3.32 34.37 ± 3.55 35.30 ± 4.25 34.75 ± 3.68
Figure 5.
 
Effect of ALK1 on the formation of TLSs. TLS formation in HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector control, (C) ALK1 RNAi, or (D) pSIREN vector. Images were taken at day 3 after transfection. The TLS counts were as follows: 7.67 ± 1.21 for pcDNA3.1+ALK1-transfected HRCECs and 15.33 ± 2.81 for the pcDNA3.1 vector control group (n = 6; P = 0.001). Cells transfected with ALK1 RNAi (C) did not form tubelike structures. The TLS count was 12.54 ± 2.37 for HRCECs transfected with pSIREN vector. Arrows: TLSs. Magnification, ×100.
Figure 5.
 
Effect of ALK1 on the formation of TLSs. TLS formation in HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector control, (C) ALK1 RNAi, or (D) pSIREN vector. Images were taken at day 3 after transfection. The TLS counts were as follows: 7.67 ± 1.21 for pcDNA3.1+ALK1-transfected HRCECs and 15.33 ± 2.81 for the pcDNA3.1 vector control group (n = 6; P = 0.001). Cells transfected with ALK1 RNAi (C) did not form tubelike structures. The TLS count was 12.54 ± 2.37 for HRCECs transfected with pSIREN vector. Arrows: TLSs. Magnification, ×100.
Figure 6.
 
Overview of the role of ALK1 in different stages of endothelial cell development. Enhanced levels of ALK1 inhibit ALK5 and stimulate the Smad1/5 pathway. The latter has no significant effect on ANG2 and VEGF (with low expression) in HRCECs that are under normal conditions. This, together with the fact that TGF-β and the Smad2/3 pathway inhibit the formation of endothelin-1, leads to endothelial cells that exhibit limited proliferation and migration. In contrast, the Smad1/5-mediated inhibition of ANG2 and VEGF (with high expression) in HRCECs that are activated (e.g., stimulated by VEGF) affects endothelial cell proliferation and migration.
Figure 6.
 
Overview of the role of ALK1 in different stages of endothelial cell development. Enhanced levels of ALK1 inhibit ALK5 and stimulate the Smad1/5 pathway. The latter has no significant effect on ANG2 and VEGF (with low expression) in HRCECs that are under normal conditions. This, together with the fact that TGF-β and the Smad2/3 pathway inhibit the formation of endothelin-1, leads to endothelial cells that exhibit limited proliferation and migration. In contrast, the Smad1/5-mediated inhibition of ANG2 and VEGF (with high expression) in HRCECs that are activated (e.g., stimulated by VEGF) affects endothelial cell proliferation and migration.
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Figure 1.
 
In vitro identification of VEGF-stimulated HRCECs. (A) Immunolocalization of LDL receptors represented by red fluorescent signal in the cytoplasm. (B) Immunolocalization of factor VIII antigen (von Willebrand Factor) represented by green fluorescent signal in the cytoplasm. The blue fluorescent regions indicate nuclei that were stained with Hoechst 33258. Magnification, ×200.
Figure 1.
 
In vitro identification of VEGF-stimulated HRCECs. (A) Immunolocalization of LDL receptors represented by red fluorescent signal in the cytoplasm. (B) Immunolocalization of factor VIII antigen (von Willebrand Factor) represented by green fluorescent signal in the cytoplasm. The blue fluorescent regions indicate nuclei that were stained with Hoechst 33258. Magnification, ×200.
Figure 2.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in pcDNA3.1+ALK1 transfected HRCECs and control cells under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 2 ).
Figure 2.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in pcDNA3.1+ALK1 transfected HRCECs and control cells under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 2 ).
Figure 3.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in ALK1 RNAi–transfected HRCECs and pSIREN vector under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 3 ).
Figure 3.
 
Evaluation of the level of (A) ALK1, (B) occludin, (C) ANG2, and (D) ALK5 in ALK1 RNAi–transfected HRCECs and pSIREN vector under stimulation of VEGF. The result of a representative Western blot experiment is shown (n = 6). The band optical density was measured by densitometry (data presented in Table 3 ).
Figure 4.
 
Evaluation of the effect of ALK1 on the migration of VEGF-stimulated HRCECs. HRCECs were transfected with pcDNA3.1+ALK1, pSIREN-ALK1 RNAi, or vector control, and the cell migration was assessed with the Boyden chamber assay. Cells were fixed in 3.7% formaldehyde and stained with HE. Migration of HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector, (C) pSIREN-ALK1 RNAi, or (D) pSIREN vector. The number of migrating cells in the groups was 12.50 ± 1.87, 23.83 ± 4.31, 37.67 ± 4.68, and 26.46 ± 2.39 (P < 0.001), respectively.
Figure 4.
 
Evaluation of the effect of ALK1 on the migration of VEGF-stimulated HRCECs. HRCECs were transfected with pcDNA3.1+ALK1, pSIREN-ALK1 RNAi, or vector control, and the cell migration was assessed with the Boyden chamber assay. Cells were fixed in 3.7% formaldehyde and stained with HE. Migration of HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector, (C) pSIREN-ALK1 RNAi, or (D) pSIREN vector. The number of migrating cells in the groups was 12.50 ± 1.87, 23.83 ± 4.31, 37.67 ± 4.68, and 26.46 ± 2.39 (P < 0.001), respectively.
Figure 5.
 
Effect of ALK1 on the formation of TLSs. TLS formation in HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector control, (C) ALK1 RNAi, or (D) pSIREN vector. Images were taken at day 3 after transfection. The TLS counts were as follows: 7.67 ± 1.21 for pcDNA3.1+ALK1-transfected HRCECs and 15.33 ± 2.81 for the pcDNA3.1 vector control group (n = 6; P = 0.001). Cells transfected with ALK1 RNAi (C) did not form tubelike structures. The TLS count was 12.54 ± 2.37 for HRCECs transfected with pSIREN vector. Arrows: TLSs. Magnification, ×100.
Figure 5.
 
Effect of ALK1 on the formation of TLSs. TLS formation in HRCECs transfected with (A) pcDNA3.1+ALK1, (B) pcDNA3.1 vector control, (C) ALK1 RNAi, or (D) pSIREN vector. Images were taken at day 3 after transfection. The TLS counts were as follows: 7.67 ± 1.21 for pcDNA3.1+ALK1-transfected HRCECs and 15.33 ± 2.81 for the pcDNA3.1 vector control group (n = 6; P = 0.001). Cells transfected with ALK1 RNAi (C) did not form tubelike structures. The TLS count was 12.54 ± 2.37 for HRCECs transfected with pSIREN vector. Arrows: TLSs. Magnification, ×100.
Figure 6.
 
Overview of the role of ALK1 in different stages of endothelial cell development. Enhanced levels of ALK1 inhibit ALK5 and stimulate the Smad1/5 pathway. The latter has no significant effect on ANG2 and VEGF (with low expression) in HRCECs that are under normal conditions. This, together with the fact that TGF-β and the Smad2/3 pathway inhibit the formation of endothelin-1, leads to endothelial cells that exhibit limited proliferation and migration. In contrast, the Smad1/5-mediated inhibition of ANG2 and VEGF (with high expression) in HRCECs that are activated (e.g., stimulated by VEGF) affects endothelial cell proliferation and migration.
Figure 6.
 
Overview of the role of ALK1 in different stages of endothelial cell development. Enhanced levels of ALK1 inhibit ALK5 and stimulate the Smad1/5 pathway. The latter has no significant effect on ANG2 and VEGF (with low expression) in HRCECs that are under normal conditions. This, together with the fact that TGF-β and the Smad2/3 pathway inhibit the formation of endothelin-1, leads to endothelial cells that exhibit limited proliferation and migration. In contrast, the Smad1/5-mediated inhibition of ANG2 and VEGF (with high expression) in HRCECs that are activated (e.g., stimulated by VEGF) affects endothelial cell proliferation and migration.
Table 1.
 
Evaluation by Real-Time PCR of ALK1, Occludin, ANG2, and ALK5 Expression in VEGF-Stimulated HRCECs That Overexpress ALK1
Table 1.
 
Evaluation by Real-Time PCR of ALK1, Occludin, ANG2, and ALK5 Expression in VEGF-Stimulated HRCECs That Overexpress ALK1
Gene Control HRCECs (n = 6) ALK1 Transfected HRCECs (n = 6) P
ALK1/β-actin 0.81 ± 0.09 1.05 ± 0.18 0.022
Occludin/β-actin 0.84 ± 0.07 1.23 ± 0.06 <0.001
ANG2/β-actin 0.74 ± 0.09 0.47 ± 0.05 <0.001
ALK5/β-actin 0.70 ± 0.03 0.42 ± 0.05 <0.001
Table 2.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2 and ALK5 Levels in VEGF-Stimulated HRCECs Producing a Higher Level of ALK1
Table 2.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2 and ALK5 Levels in VEGF-Stimulated HRCECs Producing a Higher Level of ALK1
Gene Control HRCECs (n = 6)* ALK1-Transfected HRCECs (n = 6) P
ALK1/β-actin 0.74 ± 0.08 0.90 ± 0.07 0.005
Occludin/β-actin 0.45 ± 0.08 0.77 ± 0.10 0.001
ANG2/β-actin 0.33 ± 0.03 0.16 ± 0.03 0.001
ALK5/β-actin 0.74 ± 0.07 0.43 ± 0.06 0.001
Table 3.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2, and ALK5 Levels in VEGF-Stimulated ALK RNAi Transfected HRCECs
Table 3.
 
Evaluation by Western Blot Analysis of the ALK1, Occludin, ANG2, and ALK5 Levels in VEGF-Stimulated ALK RNAi Transfected HRCECs
Gene Control HRCECs with pSIREN Vector (n = 6)* ALK1 RNAi Transfected HRCECs (n = 6) P
ALK1/β-actin 0.73 ± 0.07 0.31 ± 0.06 0.001
Occludin/β-actin 0.93 ± 0.09 0.35 ± 0.07 0.001
ANG2/β-actin 0.87 ± 0.13 1.09 ± 0.09 0.007
ALK5/β-actin 0.83 ± 0.08 1.17 ± 0.11 0.001
Table 4.
 
Effect of ALK1 on the Proliferation of VEGF-Stimulated HRCECS
Table 4.
 
Effect of ALK1 on the Proliferation of VEGF-Stimulated HRCECS
Cell Cycle Phase (%) ALK1-Transfected HRCECs (n = 6) Control HRCECs (n = 6) ALK1 RNAi HRCECs (n = 6) pSIREN Vector HRCECs (n = 6)
G0/G1 61.37 ± 2.25 40.21 ± 3.59 28.52 ± 2.32 42.62 ± 3.11
S 15.46 ± 2.08 25.42 ± 3.84 36.18 ± 3.60 23.05 ± 3.16
G2+M 23.17 ± 3.32 34.37 ± 3.55 35.30 ± 4.25 34.75 ± 3.68
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