Nirbhai Singh

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization.

Purpose: To determine whether RNA interference (RNAi) could block hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) in human corneal epithelial cells in vitro and inhibit and regress injury-induced murine corneal neovascularization in vivo. Methods: siRNA selected on the basis of target sequence homology between mouse and human VEGF was placed into expression cassettes and transfected into human corneal epithelial cells. Hypoxia-induced VEGF synthesis was assayed. Also, the effect of a plasmid capable of directing the expression of an siRNA against VEGF when injected into mouse corneas 8 hours before alkali-mechanical trauma was studied. Leukocyte count, VEGF protein levels, and degree of neovascularization in corneas were compared with that of a control siRNA plasmid. Plasmids were injected 1 week after injury to assess the ability of RNAi to regress corneal neovascularization. Results: Hypoxia-induced VEGF mRNA synthesis and protein secretion by human corneal epithelial cells was efficiently suppressed by an siRNA targeted against a sequence uniquely identical for the mouse and human VEGF genes. Intrastromal delivery of a plasmid expressing this siRNA before murine corneal injury suppressed corneal VEGF by 55.7% versus control (P = 0.014), leukocyte infiltration by 69.5% (P < 0.001), and neovascularization 1 week after injury by 72.3% (P = 0.001). At the regression time point, treated corneas had 72.8% less neovascularization (P < 0.001). Conclusions: RNAi significantly suppresses expression of VEGF induced by hypoxia in human corneal epithelial cells in vitro. In vivo, intrastromal delivery of a plasmid expressing siRNA against VEGF suppresses injury-induced VEGF expression, leukocyte infiltration, and angiogenesis and was able to regress corneal neovascularization.

Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications

There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications.

Soluble vascular endothelial growth factor receptor-1 contributes to the corneal antiangiogenic barrier.

Purpose: Pathological neovascularisation within the normally avascular cornea is a serious event that can interfere with normal vision. Upregulation of vascular endothelial growth factor (VEGF) has been associated with neovascularisation in the eye, suggesting that maintaining low levels of VEGF is important for corneal avascularity and intact vision. This study aims to determine the expression profile and possible contribution of sVEGFR-1 to the corneal avascular barrier. Design: Experimental case series investigating VEGF and soluble fms-like tyrosine kinase (sFlt) levels in normal and neovascularised human corneas. Participants: Four normal human corneas, five human corneas with alkali burns, three human corneas with aniridia, one with ocular cicatricial pemphigoid and two with interstitial keratitis were examined. Methods: Western blot and immunohistochemical analyses were performed to determine sFlt and VEGF levels in normal and neovascularised human corneas. Immunoprecipitation was utilised to demonstrate sFlt–VEGF binding. Results: Normal human corneas strongly express sFlt in the corneal epithelium and weakly in the corneal stroma close to the limbus. VEGF is bound by sFlt in the normal human cornea. Neovascularised human corneas have greatly reduced expression of sFlt and significantly less VEGF bound by sFlt. Conclusions: sFlt is highly expressed in the human cornea and normally sequesters VEGF.

Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1

Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI: http://dx.doi.org/10.7554/eLife.00324.001

Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity

Corneal transparency is a prerequisite for optimal vision and in turn relies on an absence of blood and lymphatic vessels, which is remarkable given the corneas proximity to vascularized tissues. Membrane-bound vascular endothelial growth factor receptor 3 (VEGFR-3), with its cognate ligand vascular endothelial growth factor C (VEGF-C), is a major mediator of lymphangiogenesis. Here, we demonstrate that the cornea expresses a novel truncated isoform of this molecule, soluble VEGFR-3 (sVEGFR-3), which is critical for corneal alymphaticity, by sequestering VEGF-C. sVEGFR-3 binds and sequesters VEGF-C, thereby blocking signaling through VEGFR-3 and suppressing lymphangiogenesis induced by VEGF-C. sVEGFR-3 knockdown leads to lymphangiogenesis and hemangiogenesis in the mouse cornea, while overexpression of sVEGFR-3 inhibits lymphangiogenesis and hemangiogenesis in a murine suture injury model. Pax6(+/-) mice spontaneously develop corneal and lymphatic vessels and are deficient in sVEGFR-3. sVEGFR-3 suppresses hemangiogenesis by blocking VEGF-C-induced phosphorylation of VEGFR-2. Overexpression of sVEGFR-3 leads to a 5-fold increase in corneal transplant survival in mouse models. sVEGFR-3 holds promise as a molecule to control and regress lymphatic-vessel-based dysfunction. Therefore, sVEGFR-3 has the potential to protect the injured cornea from opacification secondary to infection, inflammation, or transplant rejection.

Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration

Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.

Corneal stroma PDGF blockade and myofibroblast development.

Myofibroblast development and haze generation in the corneal stroma is mediated by cytokines, including transforming growth factor-beta (TGF-beta), and possibly other cytokines. This study examined the effects of stromal PDGF-beta blockade on the development of myofibroblasts in response to -9.0 diopter photorefractive keratectomy in the rabbit. Rabbits that had haze generating photorefractive keratectomy (PRK, for 9 diopters of myopia) in one eye were divided into three different groups: stromal application of plasmid pCMV.PDGFRB.23KDEL expressing a subunit of PDGF receptor b (domains 2-3, which bind PDGF-B), stromal application of empty plasmid pCMV, or stromal application of balanced salt solution (BSS). The plasmids (at a concentration 1000ng/microl) or BSS was applied to the exposed stroma immediately after surgery and every 24h for 4-5 days until the epithelium healed. The group treated with pCMV.PDGFRB.23KDEL showed lower alphaSMA+ myofibroblast density in the anterior stroma compared to either control group (P<or=0.001). Although there was also lower corneal haze at the slit lamp at one month after surgery, the difference in haze after PDGF-B blockade was not statistically significant compared to either control group. Stromal PDGF-B blockade during the early postoperative period following PRK decreases stromal alphaSMA+ myofibroblast generation. PDGF is an important modulator of myofibroblast development in the cornea.

Nanoparticle-Mediated Delivery of shRNA.VEGF-A Plasmids Regresses Corneal Neovascularization

PURPOSE To determine the efficacy of a plasmid containing a small hairpin RNA expression cassette (pSEC.shRNA) against VEGF-A-loaded poly(lactic co-glycolic acid) nanoparticles (PLGA NPs) in the sustained regression of murine corneal neovascularization. METHODS PLGA nanoparticles were loaded with pSEC.shRNA.VEGF-A plasmids using the double emulsion-solvent evaporation method. KNV was induced in BALB/c mice by mechanical-alkali injury. Four weeks after induction of KNV, the mice were randomly divided to receive one of four treatments intrastromally: pSEC.shRNA.VEGF-A PLGA NPs (2 μg plasmid); naked pSEC.shRNA.VEGF-A plasmid only (2 μg plasmid); control blank PLGA NPs (equivalent dry weight of NPs); and vehicle. Two and five days after intervention, corneas were harvested to determine VEGF-A gene and protein expression using reverse transcriptase polymerase chain reaction and ELISA, respectively. Four weeks after intervention, corneas were photographed, mice sacrificed, and the corneal whole mounts were immunostained for CD31 (panendothelial cell marker). Immunofluorescence microscopy was performed and the neovascular area was quantitated. RESULTS VEGF-A mRNA (49.6 ± 12.4 vs. 82.9 ± 6.0%, P < 0.01) and protein (4.0 ± 5.2 vs. 20.0 ± 7.5 ρg VEGF-A/mg total protein, P < 0.05) expression were significantly reduced in pSEC.shRNA.VEGF-A PLGA NP-treated corneas as compared with control blank NP. The pSEC.shRNA.VEGF-A PLGA NP-treated corneas showed significant regression in the mean fractional areas of KNV (0.125 ± 0.042; 12.5%, P <0.01) compared with both naked plasmid only (0.283 ± 0.004; 28.3%) and control (blank NPs = 0.555 ± 0.072, 55.5%) at 4 weeks post-treatment. CONCLUSIONS The pSEC.shRNA.VEGF-A-loaded PLGA NPs are an effective, nonviral, nontoxic, and sustainable form of gene therapy for the regression of murine KNV.

Effect of TGFβ and PDGF-B blockade on corneal myofibroblast development in mice

The purpose of this study was to investigate the role of transforming growth factor beta (TGFβ) and/or platelet-derived growth factor-B (PDGF-B) blockade on the differentiation of vimentin and alpha-smooth muscle actin (αSMA)-expressing myofibroblasts associated with haze in mice. Mouse corneas had haze-generating irregular PTK (phototherapeutic keratectomy) and topical treatment with the vectors. Six study groups of PTK treated corneas, with four corneas per group in each experiment, were Group 1) treated with TGFβ-KDEL vector interfering with TGFβ signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 2) treated with PDGF-B-KDEL vector interfering with PDGF signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 3) treated with both TGFβ-KDEL vector and PDGF-B-KDEL vector to interfere with signaling of both cytokines; Group 4) empty pGFPC1 vector; Group 5) empty pCMV vector; and Group 6) no vector treatment control. At one month after surgery, the corneas were analyzed by immunocytochemistry (IHC) for central stromal cells expressing myofibroblast markers vimentin and αSMA. The stroma of corneas treated with the TGFβ-KDEL vector alone (p < 0.05) or both the TGFβ-KDEL and PDGF-B-KDEL vectors (P < 0.05) had significantly lower density of vimentin-positive cells compared to the corresponding control group. The central stroma of corneas treated with the TGFβ-KDEL vector (p < 0.05) or the PDGF-B-KDEL vector (p < 0.05) had lower density of αSMA-positive cells compared to the corresponding control group. The density of αSMA-positive stromal cells was also significantly lower (p < 0.05) when both the TGFβ-KDEL and PDGF-B-KDEL and vectors were applied together compared to the corresponding control groups. This study provides in situ evidence that TGFβ and PDGF-B have important roles in modulating myofibroblast generation in the mouse cornea after haze-associated injury.