It is known that retinal neovascularization and fibrotic membranes are hallmarks of PDR. Anti-VEGF drugs have gradually become an effective way to assist the treatment of PDR due to their significant effect on neovascular and exudative lesions . However, a large number of studies have found that anti-VEGF drug therapy can lead to increased expression of fibrotic factors and aggravate the fibrotic process [19, 20]. Previous studies have found that CTGF expression in the proliferative membrane of PDR patients treated with anti-VEGF drugs was significantly decreased, but fibrotic factor expression was significantly upregulated . Therefore, we also hope to explore novel dual-target therapies for DR by mining its pathogenesis.
In the present study, we used RF/6A cells stimulated with high glucose levels to simulate the ocular environment in DR patients to some extent. Stimulation with high glucose levels could promote the proliferation and migration of cells, indicating the remarkable influence of a high glucose state on the biological characteristics of RF/6A cells. To further understand the mechanism by which high glucose levels affect vascular endothelial cells, we used RNA-seq to comprehensively sequence transcripts in a DR cell model. With the accurate and quantitative recognition of molecular markers by RNA-seq, we successfully identified two differentially expressed genes, BMP4 and SMAD9, which belong to the TGF-β pathway.
Bone morphogenetic proteins (BMPs), members of the TGF-β family, are involved in many cellular functions. BMP4, a member of the BMP family, is involved in eye development, including protecting Müller glial cells in the chicken retina, promoting cell invasion and migration in malignant melanoma and smooth muscle, regulating the transcription and secretion of the VEGF gene in ARPE-19 cells and zebrafish embryos, and contributing to renal fibrosis[23–28]. In detail, BMP4 increased VEGF secretion in a dose- and time-dependent manner by binding to BMP-activated SMAD-binding elements, affecting the phosphorylation of R-SMADs (SMAD1, SMAD5, and SMAD9) and forming complexes with Smad4, which ultimately activate BMP target genes [29–32]. BMP4 affects the phosphorylation of SMAD9 and forms complexes with Smad4, which finally induces BMP target genes. Many studies have shown the upregulation of SMAD9 expression by BMP4 in many cell types, such as C2C12, H9c2, 3T3-L1, HepG2, B16 cells and primary fibroblasts [33, 34]. Moreover, BMP4 significantly increased the collagen production of fibroblasts and induced the differentiation of fibroblasts into myofibroblasts . In a variety of cells, high expression of BMP4 significantly upregulates extracellular components, including FN, PAI-1, collagen, and platelet-reactive protein 1. Therefore, BMP4 and SMAD9 may function as partners to play a role in angiogenesis and fibrosis in DR.
Binding of BMP4 to the TGF-β receptor initiates SMAD9 phosphorylation and translocation into the nucleus from the cytoplasm. Afterwards, a functional protein complex consisting of SMAD4 and SMAD9 is formed, which leads to up-regulate the transcription and secretion of the VEGF gene and the promotion of extracellular matrix formation.
Considering that RNA-seq may contain false-positive results, in order to minimize this possibility, we tried our best to confirm the expression of the identified differential genes by establishing multiple DR models in diverse cell types. We first measured the expression of these two genes in the blood of people with diabetes. In DR, blood is the most practical and clinically significant experimental sample. The expression of the two factors in patient blood samples was measured to fully represent the true distribution of these two factors in DR patients. We collected blood from 20 diabetic patients and 20 healthy subjects and isolated monocytes. In the RNA extracted from peripheral blood lymphocytes, we detected the expression of these two genes by PCR. We studied the expression level of these two genes in the 20 patients on average and in each diabetic patient. The results showed that the expression of BMP4 and SMAD9 was increased in peripheral blood lymphocytes of diabetic patients, and the expression was increased in each diabetic patient.
Then, we detected the expression of these two genes in STZ-induced rat retinas. Since these two genes are highly expressed in DR, it is necessary and meaningful to detect the expression and distribution of these two genes in retinal samples. However, since it is difficult to obtain human retinal samples, we chose rat retinas as the most genetically similar experimental model available to us. The immunofluorescence results on rat retinas demonstrated that BMP4 and SMAD9 are highly expressed in all layers of the retina. Then, we extracted RNA and protein from the rat retinas, and the results showed that the expression of BMP4 and SMAD9 was upregulated at both the RNA and protein levels.
By studying the expression of BMP4 and SMAD9 in the blood of DR patients and in rat retinas, we adopted a comprehensive approach to understand the distribution of these molecules. The abovementioned results demonstrate that DR is a disease resulting from multiple factors that affects multiple cells. Subsequently, we designed a series of assays to verify the expression of BMP4 and SMAD9 in diverse disease models with multiple cell types. Therefore, considering the involvement of the neurovascular unit, we studied the expression of BMP4 and SMAD9 in HRCEC, Müller and RPE cell models, which are the representative cellular components of the neurovascular unit. DR is a multifactorial disease, and classical theories suggest that it is caused by the accumulation of glycosylated end products, lipid metabolism disorders, oxidative stress and so on. Therefore, in this study, we used five different methods to stimulate cells to mimic the microenvironment of retinal cells in DR from different perspectives. Oxidative stress is associated with DR, so we used H2O2 to stimulate cells . The effects of hypoxia and CoCl2 represent the physical and chemical characteristics of ischemic hypoxia in DR [37, 38]. In addition, 4HNE was used to simulate the accumulation of lipids during the DR process, and AGEs were used to simulate the accumulation of glycosylated end products in the DR process [39, 40]. We first used cell viability, migration and lumen formation assays to observe the effects of these stimuli on cell proliferation, migration and other biological characteristics and to determine whether each cell model was successfully established. Then, we examined the BMP4 and SMAD9 expression in these cell models, and the results showed that the increased expression of these two genes in the different cell models was consistent with the RNA-seq results.
We also stimulated different retinal cells (RF/6A, HRCEC, Müller and RPE) with BMP4 to observe the effect of BMP4 on multiple retinal cells. We found that BMP4 significantly affected the proliferation, migration and lumen formation of a variety of retinal cells. Moreover, we also observed the regulatory effect of BMP4 on SMAD9 and the effect of these two factors on the cellular functions of multiple retinal cells. From the experimental results, we found that after treating cells with BMP4, the expression of SMAD9 was significantly increased at both the RNA and protein levels. Under the condition of high levels of both BMP4 and SMAD9, the expression of fibrosis-related factors and VEGF in cells was significantly upregulated, which further confirmed our previous hypothesis. Therefore, BMP4 and SMAD9 may function together to play a role in angiogenesis and fibrosis in DR.
In recent years, a consensus has been reached in the field of ophthalmology, whereby the inhibition of the formation of new blood vessels and the control of fibrosis are considered equally important in the treatment of DR, and some pioneers have proposed the idea of dual-target therapy and have exerted considerable efforts in putting it into practice. They used VEGF inhibitors and anti-CTGF shRNA simultaneously to treat diabetic rats and found that the therapy could help restore normal transcription levels of CTGF and VEGF and improve retinal vascular dysfunction. In comparison, BMP4, which is the focus of our study, is capable of killing two birds with one stone, namely, regulating the expression of CTGF and VEGF simultaneously. Another significant advantage is that the factors related to fibrosis affected by BMP4 are far more than just CTGF, FN, and laminin α. Both SMA and collagen I are also under its control.