Co-culture of Retinal and Endothelial Cells Results in the Modulation of Genes Critical to Retinal Neovascularization
© Kumar et al; licensee BioMed Central Ltd. 2011
Received: 8 August 2011
Accepted: 23 November 2011
Published: 23 November 2011
The Erratum to this article has been published in Vascular Cell 2012 4:6
Neovascularization (angiogenesis) is a multistep process, controlled by opposing regulatory factors, which plays a crucial role in several ocular diseases. It often results in vitreous hemorrhage, retinal detachment, neovascularization glaucoma and subsequent vision loss. Hypoxia is considered to be one of the key factors to trigger angiogenesis by inducing angiogenic factors (like VEGF) and their receptors mediated by hypoxia inducible factor-1 (HIF-1α) a critical transcriptional factor. Another factor, nuclear factor kappa B (NFκB) also regulates many of the genes required for neovascularization, and can also be activated by hypoxia. The aim of this study was to elucidate the mechanism of interaction between HRPC and HUVEC that modulates a neovascularization response.
Human retinal progenitor cells (HRPC) and human umbilical vein endothelial cells (HUVEC) were cultured/co-cultured under normoxia (control) (20% O2) or hypoxia (1% O2) condition for 24 hr. Controls were monolayer cultures of each cell type maintained alone. We examined the secretion of VEGF by ELISA and influence of conditioned media on blood vessel growth (capillary-like structures) via an angiogenesis assay. Total RNA and protein were extracted from the HRPC and HUVEC (cultured and co-cultured) and analyzed for the expression of VEGF, VEGFR-2, NFκB and HIF-1α by RT-PCR and Western blotting. The cellular localization of NFκB and HIF-1α were studied by immunofluorescence and Western blotting.
We found that hypoxia increased exogenous VEGF expression 4-fold in HRPC with a further 2-fold increase when cultured with HUVEC. Additionally, we found that hypoxia induced the expression of the VEGF receptor (VEGFR-2) for HRPC co-cultured with HUVEC. Hypoxia treatment significantly enhanced (8- to 10-fold higher than normoxia controls) VEGF secretion into media whether cells were cultured alone or in a co-culture. Also, hypoxia was found to result in a 3- and 2-fold increase in NFκB and HIF-1α mRNA expression by HRPC and a 4- and 6-fold increase in NFκB and HIF-1α protein by co-cultures, whether non-contacting or contacting.
Treatment of HRPC cells with hypoxic HUVEC-CM activated and promoted the translocation of NFκB and HIF-1α to the nuclear compartment. This finding was subsequently confirmed by finding that hypoxic HUVEC-CM resulted in higher expression of NFκB and HIF-1α in the nuclear fraction of HRPC and corresponding decrease in cytoplasmic NFκB and HIF-1α. Lastly, hypoxic conditioned media induced a greater formation of capillary-like structures (angiogenic response) compared to control conditioned media. This effect was attenuated by exogenous anti-human VEGF antibody, suggesting that VEGF was the primary factor in the hypoxic conditioned media responsible for the angiogenic response.
These findings suggest that intercellular communications between HRPC and HUVEC lead to the modulation of expression of transcription factors associated with the production of pro-angiogenic factors under hypoxic conditions, which are necessary for an enhanced neovascular response. Our data suggest that the hypoxia treatment results in the up-regulation of both mRNA and protein expression for VEGF and VEGFR-2 through the translocation of NFκB and HIF-1α into the nucleus, and results in enhanced HRPC-induced neovascularization. Hence, a better understanding of the underlying mechanism for these interactions might open perspectives for future retinal neovascularization therapy.
KeywordsNeovascularization Human retinal progenitor cells (HRPC) Human umbilical vein endothelial cells (HUVEC) Hypoxia, Vascular endothelial growth factor Conditioned medium Co-culture
Neovascularization (angiogenesis) is defined as the formation of new blood vessels by sprouting of endothelial cells from pre-existing vessels. This is a multistep process, which is controlled by opposing regulatory factors and involves endothelial cells migration, proliferation, degradation of the underlying basement membrane, and assembly into tubes [1, 2]. Neovascularization plays a crucial role in several ocular diseases, including age-related macular degeneration, retinopathy of prematurity and proliferative diabetic retinopathy, which are major causes of blindness [3–6]. It often results in vitreous hemorrhage, retinal detachment, neovascularization glaucoma and subsequent vision loss. It is believed that tissue damage can stimulate release of angiogenic factors resulting in capillary proliferation [7–9]. Neovascularization is also essential for tissue repair, fetal development, and the female reproductive cycle. Changes in the regulatory factors, e.g., VEGF, may be directly related to pathological retinal diseases. These mediators can stimulate neovascularization directly by interacting with receptors on the endothelial cell surface, or indirectly by attracting and activating accessory cells.
Hypoxia is considered to be one of the key factors triggering angiogenesis by inducing angiogenic factors (like VEGF) and their receptors [10–12]. A number of studies [13–15] have shown that hypoxia plays a major role in triggering ocular neovascularization by inducing several angiogenic factors (e.g., vascular endothelia growth factors (VEGF) fibroblast growth factor (FGF), platelet-derived growth factor, (PDGF) and several others. VEGF is a 45 kDa glycoprotein and six isoforms of human VEGF have been identified that differ in their tissue specific expression pattern as well as in their biochemical and biological properties. VEGF as an angiogenic factor, multiple downstream signaling pathways have been implicated in the modulation of VEGF-dependent effects, including the regulation of cell survival, gene expression, cell proliferation and cell migration [16, 17]. However, all isoforms will bind the two VEGF receptors [18, 19]. Both receptors, VEGF receptor-1 (VEGFR-1 or Flt-1) and VEGFR-2 (or Flk-1/KDR) exhibit tyrosine-kinase activity (RTK).
Adaptation to hypoxia involves appropriate alterations in the expression of a number of angiogenesis-responsive genes. Hypoxia-induced genes are regulated by a critical transcriptional factor, namely hypoxia inducible factor-1 (HIF-1α) . HIF-1α is a widely expressed heterodimeric protein composed of HIF-1α and ARNT subunits, both of which belong to the basic helix-loop-helix PAS family . HIF-1α is a heterodimeric transcriptional factor that activates the transcription of several genes in response to low oxygen tension, including erythropoietin, transferrin, inducible nitric oxide synthase and VEGF. HIF-1α binds to DNA motifs hypoxia response elements (HREs) which have been found in the isoforms of a variety of genes that are overexpressed during neovascularization. Another factor, nuclear factor kappa B (NFκB) regulates the transcription of many genes required for neovascularization, cells adhesion, differentiation, proliferation and apoptosis [21–23]. Hypoxia can activate NFκB, which in turn, regulates the multiple human pathologies, including retinal neovascularization, diabetic retinopathy, inflammatory diseases, atherosclerosis and cancer. Hypoxia-mediated NFκB activation initiates a pathway that involves phosphorylation and subsequent degradation of the NFκB inhibitor (IKB) resulting in the cytoplasmic release and nuclear translocation of NFκB.
Many angiogenic factors are also implicated in neovascularization in the eye, where the source could be retina, pericytes, EC, or RPE. It has been suggested that an imbalance of growth factors may have a pathogenic role in retinal neovascularization. Additionally, hypoxia has been shown to induce the expression of VEGF in matured retinal cells [24–29]. To the best of our knowledge, this is the first study with HRPC co-cultured with HUVEC to examine as a model of retinal neovascularization. Our use of the HRPC cells as a model of retinal neovascularization was based on the fact that HRPCs represent only about 0.2% of pigmented cells in the ciliary margin zone, and has been shown to be multipotent, proliferative and express the neuroectodermal marker nestin . HRPC can differentiate into retinal neurons and Mûller glia, including photoreceptors . The aim of this study was to elucidate the mechanism of interaction between HRPC and HUVEC that modulates a neovascularization response. We established a trans-well co-culture system of endothelial cells (HUVEC) and HRPC under normoxia and hypoxia conditioned, and evaluated the effect of these environments as well as conditioned media from normoxia or hypoxia obtained from both cells on the angiogenic response. Further, we also studied the role of HUVEC-CM on the translocation of the transcriptional factors NFκB and HIF-1α in the HRPC cells. We found that hypoxia can affect both HUVEC and HRPC cells' gene expression independently. The factors are released into the media and can modulate endothelial-retinal cell interactions, which may lead to a number of neovascularization disorders, including retinal diseases.
Human retinal progenitor cells (HRPC) and umbilical vein endothelial cell (HUVEC) propagation
The human retinal progenitor cells (HRPC) were characterized by and obtained from Dr. Kamla Dutt [30–32]. Cells were maintained routinely in Dulbecco Modified Eagle Medium (DMEM): nutrient mixture F-12 (1:1); DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 0.075% sodium bicarbonate. The cells cultured were maintained at 37°C in a humidified air/CO2 (95:5, v/v) atmosphere and used 42-45 passages. At confluence, the media were replaced with serum-free 24 h before; cells were used for co-culture or exposed to normoxia or hypoxia conditions.
Human umbilical vein endothelial cells (HUVEC) were obtained as primary cultures from Cambrex Biosciences (San Diego, CA). HUVEC (passages 3-6) were cultured routinely in EMG-2 Bullet kit composed of endothelial cell basal medium-2 (EBM-2 medium) supplemented with 2% fetal bovine serum, human VEGF, human epidermal growth factor (hEGF), insulin-like growth factor-1, and ascorbic acid, gentamicin sulfate hydrocortisone, and heparin, as described by the manufacturer protocol . When HUVEC were cultured in co-culture with HRPC or exposed with hypoxia, cells were maintained in serum free EMG-2 medium without any growth factor for 24 h.
Co-culture of HRPC and HUVEC in transwell systems
The HRPC and HUVEC cells are grown in the non-contacting co-culture system for 24 h, which allow bidirectional diffusion of soluble factors. Cultures were serum and growth factor deprived for 24 h before the normoxia/hypoxia environment studies were initiated. All cultures were 80-85% confluent by visual inspection on the day that hypoxia experiments were initiated. The second set of the cell-cell interactions were examined between HRPC and HUVEC using contacting co-culture model maintained in transwell plates (Figure 1B). Briefly, the HRPC were seeded on the bottom on transwell system membrane inserts and HUVEC were seeded on the top. All cultures were incubated at 37°C in a humidified air/CO2 (95:5, v/v) atmosphere for the duration of the experiment. The next day media were aspirated and replaced with fresh EMB medium without serum and growth factors for 24 h before the normoxia/hypoxia conditions. Finally, the transwell plates were placed under normoxia and hypoxia incubators for specified period of time.
Monolayer cultures were incubated in a hypoxic environment by the following protocol. Briefly, media were pre-equilibrated with a hypoxic gas mixture (1%O2, 5%CO2, and 94%N2). Cultures were replenished with the hypoxic medium, placed in the hypoxia incubator and maintained for 24 h to 48 h at 37°C. Control monolayer cultures were maintained in a normoxic environment, which refers to standard cell culture in a humidified incubator (20% O2, 5% CO2, and 75% N2). All cells maintained in normoxic condition showed no morphological changes by light microscopy. The trypan blue exclusion test of cell viability showed cells to be >98% viable, and could then be sub-cultured normally. Cells incubated under normoxia conditions from the same batch, and subsequently sub-cultured, were used as control.
Preparation of HRPC/HUVEC conditioned medium (HRPC-CM/HUVEC-CM)
Quantifying secreted VEGF protein levels by ELISA
Changes in environmental factors in the cells culture can be differentially regulated by diverse signals. Therefore, we thought it was important to determine how hypoxia and co-culture interact in the regulation of VEGF secretion. Conditioned medium were collected, as described above, for the determination of secreted VEGF levels by ELISA. VEGF was assessed using commercially available human VEGF-ELISA kit (R&D Systems, Minneapolis, MN), performed according to manufacturer's instructions and normalized to protein content (BioRad, CA). The experiments were repeated three times in triplicates and are shown as mean ± SEM. The level of VEGF is expressed as pictograms of VEGF protein per milligram protein.
Capillary-like Tube Formation Assays
The effects secreted factors from HRPC-CM under normoxia and hypoxia were examined in the formation of capillary tube-like structures by HUVEC. The matrigel assay is a frequently used method for detection of angiogenesis in vitro. Briefly, the growth factor reduced Matrigel (BD Biosciences, San Jose, CA) was added to pre-chilled 48 well culture plates and allowed to polymerize at 37° C for 2 h. HUVECs (4 × 105 cells/well) were plated in the Matrigel-coated plates and the cells cultured in HRPC-CM from normoxia and hypoxia conditioned for 24 h at 37°C with 5% CO2. To study the role of VEGF in capillary tube formation, parallel cultures were treated with 20 ng/ml neutralizing anti-VEGF antibody, which were added at the time of HUVEC seeding. VEGF (50 ng/ml) was used as the positive control. Angiogenesis (capillary cord formation) were evaluated after culturing for 24 and 48 h and resulting tube-like capillary structures were examined by using an Olympus TMS inverted phase contrast microscope. Photomicrographs were documented with an Olympus digital camera.
Evaluating the expression of angiogenic protein by Western blot
The total cellular protein was isolated and used to examine the expression of angiogenic factors such as: VEGF, VEGF receptors (VEGFR-1 and VEGFR-2), and transcriptional factors (HIF-1α and NFκB). All these proteins play a major role in regulation of the retinal neovascularization. Briefly, after normoxia/hypoxia incubation for 24 h, media were collected and cells were washed with the PBS. The total proteins were extracted from the cell layers using M-PER Mammalian Protein Extraction Reagent (Pierce; CA) containing a protease inhibitor mixture (Sigma, CA). The protein extracts were centrifuged for 15 min at 15,000 rpm at 4° C and the supernatant were saved for further analysis. Total protein concentration of each sample was determined by the Bradford method as per manufacturer protocol (BioRad, CA) with bovine serum albumin as a standard. 30 μg total cellular proteins were analyzed on SDS/PAGE under reduced conditions. Before loading, all cell lysates were added to an equal volume of Laemmli sample buffer and heated at 95°C for 5 min, and samples were resolved by SDS-PAGE. The fractionated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk (BioRad, CA) in Tris buffered saline (TBS) with 0.05% Tween-20 (TBS-T) for 1 h at RT followed by incubation for 2 h with the primary antibodies in 2% milk in TBS-T. The membrane was washed three times with TBS-T for 10 min, and then incubated with the HRP-conjugated secondary antibodies, diluted in 2% milk in TBS-T, for 1 h, followed by three washes of 15 min in TBS-T. All membranes were probed with antibodies to β-actin to correct for loading and transfer differences among samples. The membranes were visualized by an enhanced chemiluminescence (ECL) Western Blotting Analysis System (Amersham).
Evaluating the expression of angiogenic transcriptions by RT-PCR
List of Primers:
5'- GCC ATG GAC GAA CTG TTC CCC-3'
5'- GCC ATG GAC GAA CTG TTC CCC-3'
QuantumRNA Classic II (Ambion Inc. TX)
Expression and Translocation of NFκB and HIF-1α
We also examined the expression and translocation of NFκB and HIF-1α in HRPC cultured in HUVEC-CM (normoxia/hypoxia) by western blot analysis and immunofluorescence studies.
HRPC cells were cultured on 4-well chamber slides to approximately 80% confluence; then the cells were grown overnight under serum free medium. The next day, the medium was removed and cells were further cultured with HUVEC-CM (normoxia and hypoxia) at 37o C in a humidified air/CO2 (95:5, v/v) atmosphere for 24 h. Cultures were then rinsed with pre-warmed PBS and fixed with 4% paraformaldehyde (Sigma) in PBS for 10 min at room temperature. The fixed cultures were washed and permeabilized with a solution of PBS containing 0.1% Triton X-100 (sigma) in PBS. Non-specific antibody binding sites on the cells were blocked by using 10% goat serum for 1 h at room temperature before incubation with primary antibodies. Cultures were then incubated with primary antibody rabbit polyclonal antibody against NFκB and HIF-1α (Santa Cruz Biotechnology, CA) overnight at 4° C. Cells were washed and incubated with FITC-conjugated anti-rabbit IgG (1:100 dilution) for 1 h at room temperature. For nuclear counter-stain DAPI (Molecular probe; CA) was used as per manufacturer's protocol.
Cultures were incubated as described above for culture of normoxia/hypoxia HUVEC-CM-treated cells. After incubations, the medium was removed and cultures were washed with PBS. Cytoplasmic and nuclear proteins for Western blots were prepared using Nuclear Extract kits (Active Motif, Carlsbad, CA) according to manufacturer's suggested protocols. Protein concentration was determined by the Bradford method (Bio-Rad, Richmond, CA). Translocation of NFκB and HIF-1α were determined by western blotting as described above.
HRPC-CM induces neovascular responses by HUVEC
VEGF secretion by individual cultures and co-cultures
Effect of hypoxia on VEGF and VEGFR-2 expression
We also observed that hypoxia induced an increase in VEGF and VEGFR-2 protein expression following western blot analysis [Figure 5E and 5F]. Our Western blot data support mRNA data with the low protein expression levels of VEGF and VEGFR-2 in HRPC and HUVEC were cultured under normoxia environment. We have found that exogenous VEGF expression is increased in HRPC and HUVEC cultured alone under hypoxia condition as compared with normoxia. There was a four-fold increase in VEGF expression by HRPC exposed to hypoxia for 24 h, as compared with normoxia. When HRPC were cultured in presence of HUVEC under hypoxia, there was a further two-fold increase in VEGF expression, compared to HRPC cultured alone under hypoxic conditions. In addition, our data indicated that hypoxia was capable of inducing not only VEGF expression, but also its receptor - VEGFR-2 when HRPC were co-cultured with HUVEC for 24 h. Data from RT-PCR and Western blot analysis are consistent with our VEGF ELISA results, and showed an increased in VEGF in all the hypoxia treatments and co-cultures compared with normoxia controls. These results suggest the association of VEGF-VEGFR-2 signaling regulate the retinal neovascularization.
Role of hypoxia on the expression of NFκB and HIF-1α
Effect of HUVEC-CM on the Subcellular Localization of NFκB and HIF-1α
Retinal neovascularization is a complex process that is not completely understood. In retinal neovascularization, there are abnormally formed blood vessels and fibrous tissue that grow from the retina or the optic disk along the posterior surface of the vitreous. These retinal capillaries do not normally invade the vitreous or extend beyond the inner plexiform layer of the retina. However, in retinal diseases, e.g., diabetic retinopathy, the disruption of the balance between angiogenic and anti-angiogenic factors favors neovascularization, which enters the sub-retinal space, where leakage and bleeding lead to retinal detachment and photoreceptor death . Clearly, the process of retinal neovascularization is mediated by the regulation of several angiogenic inducers, inhibitors, their respective receptors, and transcriptional factors. VEGF is a potent angiogenic and permeability enhancing factor causally linked to neovascularization in cancer, wound healing, diabetic retinopathy, and age-related macular degeneration [14, 34]. Several reports have shown that VEGF is produced by several cell types within the eye (retinal pigment epithelial cells, glial cells, retinal capillary pericytes, endothelial cells, Müller cells and ganglion cells) [ 35 ] and play a role in the development of retinal neovascularization [13, 36, 7]. Aiello et al.  showed that VEGF played a significant role in mediating retinal neovascularization in diabetic patients. Using animal models of ischemic retinopathy and iris neovascularization, several researchers found that VEGF expression is upregulated in the retina during neovascularization [13, 7, 8, 38]. Furthermore, inhibiting VEGF using VEGF receptor chimeric proteins, neutralizing antibodies, and antisense oligonucleotides in these animal models could reduce both retinal and iris neovascularization. Finding increased VEGF levels during retinal neovascularization has resulted in a controversy as to whether VEGF itself is sufficient to stimulate retinal neovascularization.
Increase VEGF expression could be the result of the retina microenvironment (hypoxia or ischemia) as well as changes in other factors such as pigment epithelium-derived factor (PEDF). The balance between the expression of VEGF and pigment epithelium-derived factor (PEDF) in modulating retinal neovascularization was investigated by Zhang and co-workers . These researchers found a reciprocal interaction between VEGF and PEDF in retina; significant increases in VEGF expression was observed following silencing of the PEDF gene. Hypoxia has been shown to upregulate VEGF mRNA in retinal endothelial cells, pericytes, RPE cells, Muller cells and ganglion cells [14, 40]. Our studies show that both HUVEC and HRPC express basal levels of VEGF and VEGFR-2 mRNA and protein, which was significantly increased by hypoxia treatment. Additionally, we found that co-cultures of these cell lines resulted in a synergistic increase in the expression of both VEGF and VEGFR-2, similar to that induced in single cultures by hypoxia. In fact, co-cultures treated with hypoxia showed a pronounced increase in the expression of both VEGF and VEGFR-2. However, contacting co-culture did not show a difference in response to hypoxia treatment from that of non-contacting co-cultures. Recently, Dardix and coworkers  also investigated the effects of co-culture (with retina pigmented epithelial cells - RPE) and hypoxia on endothelial cell expression of VEGF. These researchers reported that contact co-cultures upregulated both VEGF mRNA and protein expression to the same degree as hypoxia alone. In contrast to our findings, these researchers found that non-contacting co-cultures did not show an effect. However, these researchers used RPE cells for co-cultures as well as utilized CoCl2, a hypoxia mimetic to study the effect of hypoxia. In our studies we used human retinal progenitor cells (HRPC) cells for co-cultures; these cells express and secrete VEGF, among other soluble factors those have direct effects on endothelial cells . In addition, we also used hypoxic culture conditions rather than the hypoxia mimetic, which may activate additional mechanisms not seen with the hypoxia mimetic.
Hence, our studies are consistent with the idea that hypoxia-inducing VEGF expression will lead to an enhanced neovascular response. These data not only highlight the important angiogenic properties of VEGF, but they also demonstrate the autocrine ability of VEGF to regulate the synthesis of multiple angiogenic proteins . The autocrine effect of VEGF on retinal elements previously has been reported, for both retinal glial cells [43, 44] and RPE cells , but not for the HRPC. To our knowledge, however, the regulation of VEGF receptor expression has not yet been investigated in retinal neovascularization induced by hypoxia co-culture system. Several lines of evidence indicate that enhanced VEGF expression in response to hypoxia is due to transcriptional activation [45, 46]. Furthermore, this is the first time HRPC cells have been shown to have VEGF receptors (VEGFR-2) and induced the expression after hypoxia treatment. If similar autocrine functions of VEGF were present in HRPC cells, this would explain our observation of increased expression of VEGF and VEGFR-2 receptor after hypoxia and co-cultured conditioned.
It is well-established that hypoxia will stimulate neovascularization through inducing HIF1α, which translocates into the nucleus, binds to the hypoxia-response element, and up-regulates the expression of VEGF and other angiogenic factors . Several studies have shown that hypoxia induces VEGF expression in matured retinal cells, which may play a major role in development of many retinal diseases. Our data from nuclear staining pattern and the immunoblotting of nuclear extracts indicate that HIF-1α, present in the cytoplasm of HRPC treated with normoxia HUVEC-CM, did translocate to the nucleus when cells were treated with hypoxia HUVEC-CM. HIF-1 may also have a function in retina that is unrelated to hypoxia because the constitutive level of HIF-1 expression in adult normoxic retina is much higher than that found for heart and lung, where it was undetectable by immunoblot or immunohistochemistry. VEGF is also constitutively expressed in the retina and it is possible that this is driven by the constitutive expression of HIF-1.
Another mechanism that may be activated by hypoxia treatment is NF-κB, which has also been suggested to play an important role in the induction of retinal neovascularization [21, 23]. In the present study, we found that hypoxia has increases expression of NFκB mRNA and protein in HRPC/HUVEC cultured and co-cultured conditions. The hypoxic sensitivity of the NF-κB pathway and a wide variety of in vitro studies have demonstrated that exposure of various cell types to hypoxia activates NF κB signaling pathways. There are several reports that NFκB was activated in retinal glial cells, vascular endothelial cells, pericytes, and macrophage/microglia, all of which participate in neovascular disorders such as diabetic retinopathy. In the retina, NF-κB is localized in sub-retinal membranes and in microvessels and is activated very early in the course of development of retinopathy in diabetes. The activation of NFKB in retinal pericytes is responsible for the hyperglycemia-induced accelerated loss of pericytes observed in diabetic retinopathy. An inhibitor of NF-κB, PDTC inhibited NF-KB activation and reduced retinal neovascularization without discernible toxicity in a mouse model [45, 46]. It also revealed that NF-KB might provide a basis for a specific and effective therapeutic regimen for various diseases associated with undesired angiogenesis. Normally, NFκB is present in the cytoplasm as a complex with members of the IkB inhibitor family. The hypoxia-induced activation of NFκB could result in the degradation of IkB. This in turn exposes the nuclear localization signal, allowing NFκB to be transported into the nucleus. In this study, we investigated the effects of normoxia/hypoxia exposed HUVEC-CM on NFκB nuclear translocation. Our studies showed that the HRPC cells did not significantly affect the nuclear translocation of NFκB when the cells were exposed with normoxia HUVEC-CM. However, nuclear translocation of NFκB was induced by hypoxia HUVEC-CM. Since nuclear localization of NF-κB is thought to be equivalent to NF-κB activation, we have shown that NF-κB was activated in HRPC cells by hypoxia HUVEC-CM, which may have triggered the transcription of NF-κB dependent genes and regulated the expression retinal neovascularization related genes.
Our findings suggest that direct or indirect communications between HRPC and HUVEC cells results in the expression and activation of transcription factors associated with enhanced expression of pro-angiogenic factors under hypoxic conditions. These mechanisms play necessary roles in producing an enhanced neovascular response. Furthermore, our data indicate that the hypoxia treatment results in the up-regulation of both mRNA and protein expression for VEGF, and VEGFR-2 through the translocation of NFκB and HIF-1α to the nucleus, resulting in enhance HRPC-induced neovascularization. Hence, these results have provided a better understanding of the underlying mechanism involved in hypoxia-induced retinal neovascularization, which may generate new perspectives for future retinal neovascularization therapy.
List of Abbreviations used
human retinal progenitor cells
human umbilical vein endothelial cells
vascular endothelial growth factor
vascular endothelial growth factor receptor -2
The research was supported by NIH in part by funds from RCMI-3G12RR003034-34S1.
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