Proliferative vitreoretinopathy (PVR) is a complication of a retinal detachment and occurs in approximately 8–10% of patients developing retinal detachment [
PVR can be divided into multiple categories based on the configuration of the retina and the location of the scar tissue [
Vascular endothelial growth factor A (VEGF-A), platelet-derived growth factors (PDGFs), and non-PDGFs (growth factors outside of the PDGF family) are relevant to PVR pathogenesis because of their role in suppressing p53 levels through different pathways [
The dynamic regulation of VEGF by the p53 family members makes its regulation complex, especially given the fact that all of the three transcription factors (p53, p63, and p73) are able to induce and repress VEGF, which appears to be dependent on cellular context and stimulus [
The aim of the present study is to evaluate the expression levels of VEGF-A, Otx1, Otx2, Otx3, and p53 family genes in adult healthy human retinae in comparison with retinae affected by PVR and to try to understand their role in inducing or preventing this eye pathology.
Twelve human retinae samples were taken from twelve patients affected by PVR during vitreoretinal surgery by the same operating surgeon (CA). PVR was graded according to the Retina Society Terminology Committee [
Patients’ data in PVR surgery.
Patients | Age* | Sex | Refraction (SE) | Visual acuity before surgery* | PVR stage* | Total number of vitreoretinal surgeries* | Visual acuity final outcome** |
---|---|---|---|---|---|---|---|
1 | 65 | F | −1.00 | 0.4 | C2 | 2 | 0.4 |
2 | 58 | M | −1.50 | 0.2 | C2, CA1 | 2 | 0.3 |
3 | 67 | M | −1.00 | 0.3 | C3 | 2 | 0.2 |
4 | 62 | M | +1.75 | 0.1 | C6, CA2 | 5 | 0.1 |
5 | 62 | M | +2.00 | 0.1 | C8, CA3 | 6 | 0.05 |
6 | 45 | M | −7.50 | 0.02 | C7 | 2 | HM |
7 | 80 | M | +1.00 | HM | C6 | 4 | HM |
8 | 70 | F | 0 | 0.02 | C5 | 2 | HM |
9 | 72 | M | +1.00 | 0.2 | C4 | 1 | 0.2 |
10 | 51 | M | −1.00 | HM | C6 | 4 | 0.05 |
11 | 77 | M | 0 | HM | C7 | 2 | HM |
12 | 57 | M | −6.00 | HM | C11, CA4 | 2 | LP |
SE: spherical equivalent. HM: hand motion. LP: light perception. PVR: proliferative vitreoretinopathy. PVR stage: presence of preretinal or subretinal membranes posterior (C) or anterior (CA) to the equator of the eye, and number of clock hours involved (from 1 to 12). *at the time of retinectomy in PVR surgery. **six months after surgery.
Surgical procedures were similarly conducted in all patients. After accurate vitreous and vitreous base removal with vitrectomy (Stellaris, Bausch&Lomb, Rochester, New York, USA), endoilluminator and room temperature infusion fluid at surgical microscope (OPMI 1, Carl Zeiss, Jena, Germany), and accurate epiretinal membranes’ peeling with surgical instruments, it was impossible to reattach the peripheral retina because of strong epi- and intraretinal PVR tissue in peripheral retina. Therefore, peripheral retinectomy preceded by endodiathermy of adjacent retina tissue was necessary in each patient to allow final total retinal reattachment at the end of surgery (Figure
Important surgical steps. Left: peeling of epiretinal membranes using endoillumination and surgical instruments entering the eye through sclerotomies. Right: cut of peripheral retina (retinectomy) allowing further retinal reattachment.
Retina grasp. After retinectomy, a small retinal portion is grasped with nontraumatic forceps and is removed from the eye.
RNA was isolated using EuroGold Total RNA Mini Kit (Euroclone, Milan, Italy) and quantification of total RNA was performed by NanoDrop ND 1000 spectrophotometer (Thermo Scientific, Wilmington, USA). Total RNA (1
Quantitative real-time reverse transcriptase PCR (qRT-PCR) was performed by TaqMan technology using ABI Prism 7000 apparatus (Applied Biosystems, Foster City, CA, USA). Gene expression analyses were done with TaqMan Assays-on-Demand containing primers and fluorescent probe mix (Applied Biosystems, Foster City, CA, USA). PCR reaction mix contained 12.5
Statistical correlations between gene expressions were calculated with dispersion plots. We correlated two genes for graphic and values were considered significant with a linear regression coefficient
Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded sample of healthy human retina used as control. Three
To our knowledge, this is the first time that the expression of Otx genes is found in human adult retina and in PVR tissue. In the control samples, we found basal expression of all genes. In PVR samples, we found little (samples 1, 2, 3, 4, 8, 9, and 10) or no expression (samples 5, 6, 7, 11, and 12) of Otx1, attesting the nondifferentiated state of retinal tissues [
Gene expression levels in adult healthy human samples and in patients affected by PVR. Gene levels were detected by quantitative PCR.
Statistical correlation among VEGF-A, Otx2, p53, and p63 genes and between Otx1 and Otx3 genes. Dispersion plots correlate two genes for graphic.
qRT-PCR showed a correlation (
By immunohistochemical analysis we observed positivity for anti-Otx2 antibody in different retinal layers of adult healthy human sample. Photoreceptors (rods and cones), horizontal cells, bipolar and ganglion cells showed positivity for the Anti-Otx2 antibody, on the contrary of Muller cells. We confirmed the presence of Otx proteins in nucleus and cytoplasm of human retina (Figure
Histological section of healthy adult human retina. The different retinal layers appear marked in dark-marron with rabbit anti-Otx2 polyclonal antibody. Positivity is shown (from top to bottom) in photoreceptors, horizontal, bipolar, and neuronal cells (asterisks). An artifact (empty space) is present due to fixating procedure (star).
Zebrafish, like many members of the ray-finned fish (teleosts), have the innate capacity to regenerate tissues (e.g., fins, heart, and eye) [
PVR is a proliferative disease and the knowledge of expressions of some key regulatory genes could be useful to understand the disease and, hopefully, retina regeneration. The purpose of the present study was to evaluate the expression of VEGF-A, Otx1, Otx2, Otx3, p53, and p63 genes in adult healthy human retina from autopsy and in surgically removed retinae of PVR affected patients.
We confirmed the presence of VEGF-A and p53 family members and, for the first time, we found the expression of Otx genes in controls, also confirmed by immunohistochemical analysis, and in PVR tissue (Figures
Statistical analysis showed a correlation among VEGF-A, Otx2, p53, and p63 and between Otx1 and Otx3 (Figure
The two groups of genes showed reverse trends. In fact, patients with higher expressions of VEGF-A, Otx2, p53, and p63 (samples 5, 6, 7, 11, and 12) showed no expression of Otx1 and low levels of Otx3 in comparison to the other samples. On the contrary, patients showing low levels of VEGF-A, Otx2, p53, and p63 genes (samples 3 and 4) had higher levels of Otx1 and Otx3. Thus, Otx1 and Otx3 showed a significant correlation, inversely to what is observed in Otx2 expression. In fact, it is reported that Otx3 significantly suppresses Otx2-induced transcription activity, suggesting that Otx3 functions as a transcription repressor of Otx2 by acting competitively on the consensus TAATCC sequence [
In our case series, the functionality of the retinae undergoing genetic study is similar at the time of surgery and at six months followup (results shown in Table
It was demonstrated that p53 can influence VEGF-A expression both increasing and repressing its levels and that, likewise, the different isoforms of p63 have different effect on VEGF-A expression [
During standard vitreoretinal surgeries using room temperature infusion fluid, the vitrectomy cavity and retina reach deep hypothermia. After closing the infusion line, intraocular tissue rewarms rapidly [
Immunohistochemical analysis showed the presence of Otx proteins in the internal part of photoreceptors, in horizontal cells, in bipolar cells, and in neuronal cells. Despite the fact that the role of Otx1, Otx2, and Otx3 has been proved during embryo development, little is known about their functions in adult human retina. We hypothesize that Otx2 could have a function of maintenance in the identity and survival of adult retinal differentiated cells, and that its high expression in PVR patients correlates with a reentry of differentiated cells into proliferation cycle and staminal status. Precursor cells, probably in an attempt to regenerate death retina cells, on the contrary could mature in fibroblasts that produce fibrocellular membranes. Interestingly, in very severe PVR cases this process is more stimulated and it is accompanied by higher expression of Otx2.
One of the main targets of genetic studies is to translate evidence and benefits into clinical practice. In our study, we attempted to reach this objective in two different ways. Firstly, our study shows the elevated expression of VEGF-A in PVR patients, and VEGF-A was found to promote the bioactivity of vitreous in patients and rabbits with PVR. In the latter, the anti-VEGF ranibizumab injected intravitreally was found to neutralize PVR [
We found expressions of VEGF-A, p53 family, and, for the first time, Otx homeobox genes in healthy adult human retinae and in retinae affected by PVR. We individuated two possible pathways, VEGF-A, Otx2, p53, and p63 and Otx1 and Otx3 involved in PVR genes pattern that could influence in different manners the course of the pathology. In particular, in many samples of patients with more severe features of PVR, we found higher levels of VEGF-A, Otx2, p53, and p63 genes inversely to Otx1 and Otx3 expression. The anti-VEGF ranibizumab molecule, neutralizing retinal VEGF, could protect retina from PVR.
The immunohistochemical analysis of human healthy retina showed the presence of Otx proteins in many layers of the retina, confirming the hypothesis of their role in acting as a survival factor. In retinae with PVR, the highly expressed levels of Otx2 suggest the role of this gene in the proliferation of retinal stem cells as replacement for dead cells. Further studies are needed to better comprehend the genetic mechanism subtended to PVR exogenesis.
A better understanding of cellular and molecular mechanisms that regulate growth-associated induced neurogenesis in the retina may lead to new approaches for enhancing or controlling the proliferative and regenerative capacity for future therapeutic uses.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank Elisa Muscianisi of Novartis for supporting and funding the proposal and for her writing assistance in the preparation of the paper. They thank the designer Davide Bonadonna for his illustrations and the Centre of High Technology Instruments of the University of Insubria.