Ocular neovascularization caused by herpes simplex virus type 1 infection results from breakdown of binding between vascular endothelial growth factor A and its soluble receptor

Abstract

The normal cornea is transparent, which is essential for normal vision, and although the angiogenic factor vascular endothelial growth factor A (VEGF-A) is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs after ocular infection with HSV, which causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis. We demonstrate that HSV-1 infection causes increased production of VEGF-A but reduces sVR-1 levels, resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, -7, and -9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, inhibition of sVR-1 breakdown with the MMP inhibitor marimastat, and the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of stromal keratitis, an important cause of human blindness.

Figure 1. Normal cornea expresses VEGF-A bound to sVR-1

WT mice were sacrificed and 4 to 6 corneas were collected and pooled for WB, Co-immunoprecipitation, and mRNA analysis by RTPCR. Data are representative of two independent experiments. (A) Reducing WB analysis for sVR-1 and VEGF-A reveals presence of sVR-1 at 62 kDa and two isoforms of VEGF-A at 22 and 16 kDa in normal cornea. (B) Non-reducing WB for VEGF-A shows the presence of a much larger band between molecular size of 100 to 150 kDa consistent with its being the bound form. (C) sVR-1 was immunoprecipitated from normal corneal extracts, with anti sVR-1 mAb and the complex was analyzed for the co-immunoprecipitation of VEGF-A by WB. Normal cornea shows the presence of VEGF-A bound to sVR-1. (D) Agarose gel analysis of VEGF-A (214bp) (lane 3) and sVR-1 (358bp) (lane 5) cDNA from normal uninfected cornea. Lane M-marker, lane 1- β-actin (92bp), lane 2, 4 and 6 are reverse transcriptase negative controls for β- actin, VEGF-A and sVR-1. (E) Representative immunofluorescence staining of corneal section for sVR-1 (green, left panel), nuclei (blue, middle panel) reveals the presence of sVR-1 mainly in the corneal epithelial layer and to a lesser extent in the stroma (Merge). Nuclei were stained (blue) with DAPI. Image is representative of two independent experiments. Original magnification × 10.

Figure 2. Corneal HSV-1 infection causes an imbalance between VEGFA and sVR-1

WT mice corneas were scarified and infected with 10⁴ PFU of HSV in PBS or mock infected with only PBS (naïve control mice). Corneas were harvested from infected mice at indicated time points PI and from naive control mice at 24 hr. (A) At each time point 6 corneas were collected and pooled for mRNA extraction. The expression levels of VEGF-A molecule were normalized to β-actin using ΔCt calculation. Relative expression between control and infected groups were calculated using the 2^−ΔΔCt formula. Kinetic analysis for the expression of VEGF-A mRNA by quantitative PCR (QPCR) at different days pi. The expression of VEGF-A shows a biphasic upregulation pattern, early on day 2 pi and then again on day 11 pi as compared to naïve cornea. Figure is a summary of two independent experiments and each experiment represent group of 6 corneas. (B) At each time point 6 corneas were harvested from infected or naïve mice and the levels of VEGF-A were determined by ELISA. Quantification of VEGF-A protein levels in the normal and infected corneas at different days pi reveals similar pattern as that of gene expression. Figure is a summary of at least two independent experiments and each experiment represent group of 6 corneas. P ≤ 0.009 (**), P = 0.0004 (***). (C) At each indicated time points 3–4 corneas were harvested and total protein concentration were measured by BCA protein assay. Samples with equal protein concentration were subjected to SDS-PAGE followed by WB analysis. VEGF-A expression was analyzed by WB analysis showed an increase in the VEGF-A protein levels at different days pi compared to naïve control mice. (D) Three to four corneas were harvested from day 3 pi or naïve control mice at 24 hr pi. Samples with equal protein concentration were subjected to native-PAGE followed by WB analysis. The left figure shows a band of molecular size between 100 to 150 kDa in naïve corneal sample. The right is from day 3 infected corneas. It shows the large 100–150 kDa band as well as another at 50 kDa indicating the presence of both bound and free VEGF-A. (E) Kinetic analysis for the expression of sVR-1 mRNA by QPCR at different days pi. Relative change in the expression of sVR-1 mRNA at different days pi as compared to the uninfected mice revealed a sharp decrease in the sVR-1 expression from day 3 pi which reached above normal around day 11 pi and back to normal levels at day 14 pi. The figure is a summary of two independent experiments and each experiment represent group of 6 corneas. (F) sVR-1 protein levels analyzed by ELISA from the normal and infected corneas at different days pi shows an early decrease after HSV-1 infection which reaches to level found in normal controls at day 14 pi. The figure is a summary of at least two independent experiments and each experiment represent group of 6 corneas. P ≤ 0.05 (*), P = 0.0023 (**), P ≤ 0.0001 (***). (G) Quantification of total number of sVR-1 molecules per VEGF-A molecule from naïve and infected corneas at different days pi using protein quantities obtained by ELISA (B & F) shows the significant reduction of total number of sVR-1 molecules per VEGF-A molecule on day 1 and from day 7 to 14 pi. The total number of molecules for sVR-1 and VEGF-A were calculated by multiplying the total quantity of the protein by its molecular weight and Avogadro’s number. The figure is a summary of values obtained from figure B and F. P = 0.0216 (*), P ≤ 0.0032 (**), P ≤ 0.0002 (***). (H) Reducing WB analysis for sVR-1 from uninfected and infected corneas at different time pi shows degradation of sVR-1 after HSV-1 infection. Degraded products are shown by black arrowhead.

Figure 3. rsVR-1 administration hinders angiogenic activity of VEGF-A and inhibits corneal angiogenesis post HSV-1 infection

WT mice were ocularly infected by corneal scarification with 10⁴ PFU of HSV. (A) rsVR-1 or isotype Fc protein were administered subconjunctivally at 5 μg/eye as shown. The extent of angiogenesis and SK lesion severity in the eyes of HSV infected mice were quantified in a blinded manner using a scale as described in materials and methods. The progression of angiogenesis and SK lesion severity were significantly reduced in the group of mice treated with rsVR-1 as compared to isotype treated mice. Data are representative two independent experiments (n = 10–12 mice/group). P ≤ 0.0007 (***). (B) Representative whole mount corneas stained for CD31 (lower panel) at day 15 pi shows reduced angiogenic response in rsVR-1 treated mouse. Bars, 100μm. (C) Representative eye photos shows reduced SK lesion severity as well as angiogenesis from rsVR-1 treated mouse compared to isotype treated mouse. (D) rsVR-1 or isotype Fc protein treated mice were sacrifice on day 15 pi and corneas were collected for surface staining of eye infiltrating cells. Total cell numbers per cornea for CD31⁺ cells, Neutrophils (CD11b⁺, Ly6G⁺ cells) gated on total CD45⁺ cells and CD4⁺ T cells showed the significant reduction in total numbers of the respective cell population from rsVR-1 treated mice as compared to the isotype treated mice. Data are a representative of two independent experiments (n = 3–5/group). P = 0.012 (*), P ≤ 0.0015 (**). (E) Therapeutic administration of the rsVR-1 was started from day 7 pi when SK becomes visibly evident. Therapeutic administration of rsVR-1 also showed significant reduction in angiogenesis as well as lesion severity further confirming the inhibitory effect of sVR-1 on the angiogenic activity of VEGF-A. Data are a representative of two independent experiments (n = 8 mice/group). P ≤ 0.012 (*).

Figure 4. Metalloproteinases are upregulated after HSV-1 infection and MMP-2, 7 and 9 degrades rsVR-1 and sVR-1

(A) WT mice corneas were scarified and infected with 10⁴ PFU of HSV in PBS or mock infected with only PBS (naïve control mice). Corneas were harvested from infected mice at indicated time points PI and from naive control mice at 24 hr. At each time point 6 corneas were collected and pooled for mRNA extraction. The expression levels of different MMP molecule were normalized to β-actin using ΔCt calculation. Relative expression between control and infected groups were calculated using the 2^−ΔΔCt formula. Kinetic analysis for the relative fold change in the expression levels of MMP- 2, MMP-7, MMP-8, MMP-9 and MMP-12 mRNAs at different days pi. The mRNA expression of all the tested MMPs was upregulated in biphasic manner post HSV-1 infection with the first peak expression at day 1 pi and maximum expression between day 7 and day 11 pi. (B) Mouse rsVR-1 was incubated with activated form of MMP-2, MMP-7, MMP-8, MMP-9 and MMP-12 for 12 hrs at 37°C and analyzed by WB. MMP-7 (lane 3) showed maximum degradation of rsVR-1 followed by MMP-9 (lane 5) and MMP-2 (lane 2). Addition of the MMP inhibitor, EDTA, inhibited the degradation of rsVR-1 by MMP-7 (lane 7) and MMP-9 (lane 8). Figure is a representative of three independent experiments. (C) Corneal lysates from 4–6 pooled corneas of uninfected WT mice were incubated with activated form MMP-2, MMP-7, MMP-8, MMP-9 and MMP-12 either for 6 hr or 12 hr at 37°C. The digested corneal lysates were analyzed by WB for sVR-1. MMP-7 showed degradation of corneal sVR-1 even after 6 hrs of incubation (upper panel, lane 2), with complete degradation after 12 hrs of incubation (lower panel, lane 2). MMP-2 (lower panel, lane 1) and MMP-9 (Lower panel lane 4) also showed degradation of corneal sVR-1 after 12 hrs of incubation. Addition of MMP inhibitor EDTA with MMP-7 (lane 6) showed inhibition of sVR-1 degradation. Figure is a representative of three independent experiments.

Figure 5. Blocking MMPs activity in vivo rescues sVR-1 degradation and diminishes angiogenesis and SK severity

WT mice corneas were scarified and infected with 10⁴ PFU of HSV or mock infected with only PBS (naïve control mice). Marimastat, a broad-spectrum MMP inhibitor (MMPi) was administered subconjunctivally at 200 ug/mouse as shown. Mice in control group received HSV infection with mock treatment. (A) WB analysis of the corneal lysates collected from naïve (N), control treated (C) and MMPi treated mice at day 7 pi shows that MMPi rescues degradation of sVR-1 post HSV-1 infection. The figure is a representative of two independent experiments. (B) Preventive treatment of MMPi in mice infected with HSV-1 was started from day 2 pi as shown. MMPi treatment showed a significant reduction of angiogenesis as well as SK lesion severity at day 15 pi. Data are representative of two independent experiments (n = 9–12 mice/group). P ≤ 0.04 (*), P = 0.0065 (**). (C) MMPi treated or control mice were sacrifice on day 15 pi and corneas were collected for surface staining of eye infiltrating cells. Analysis for total cell numbers per cornea for CD31⁺ cells, neutrophils (CD45⁺ CD11b⁺ Ly6G⁺ cells) and CD4⁺ T cells showed significant reduction in frequencies as well as total numbers of the respective cell population in the MMPi treated mice. Data are a representative of two independent experiments (n = 7/group). P = 0.0115 (*), P = 0.0078 (**), P = 0.0002 (***). (D) Therapeutic administration of the MMPi (started from day 5 pi), also showed reduced angiogenesis as well as SK lesion severity at day 15 pi. Data are a summary of two independent experiments (n = 6 mice/group). P ≤ 0.021 (*). (E) Analysis of total cell numbers per cornea for CD31⁺ cells, neutrophils (CD45⁺CD11b⁺, Ly6G⁺ cells) and CD4⁺ T cells also showed the significant reduction in the total cell numbers per cornea after preventive MMPi treatment. Data are a representative of two independent experiments (n = 5–6/group). P ≤ 0.0094 (**).

Figure 6. Neutrophil, the principal cell in cornea after HSV-1 infection is source of MMPs and VEGF-A

WT mice corneas were scarified and infected with 10⁴ PFU of HSV or mock infected with only PBS (naïve control mice). At each indicated day pi two mice were sacrificed, corneas were harvested and single cell suspensions were analyzed for surface staining by flow cytometry. (A) Representative FACS plot from each time point shows the percentage of neutrophils (CD11b⁺Ly6G⁺) gated on total CD45⁺ cells. (B) Total number of neutrophils per cornea at each indicated time point post HSV-1 infection. Data are expressed as mean ± SEM. Figure is a summary of two independent experiments and each experiment represent group of 2 corneas. (C) Ly6G⁺ neutrophils were purified from day 2 pi corneas using anti-Ly6G beads. Cell suspension from 6–8 pooled corneas was used for neutrophil purification. Western blot analysis for MMP-2, MMP-8, MMP-9 and MMP-12 from purified neutrophils. Data is from single experiment and represent neutrophils from group 6–8 corneas. (D) Neutrophil depletion was carried out from day 7 pi, using anti-Gr-1 mAb (100 μg/mice, intra-peritoneally) as shown. Mice in the control group received isotype control antibody. Representative FACS plot shows complete depletion of neutrophils from spleen (upper panel) and cornea (lower panel) analyzed at day 11 pi. (E) Corneal lysates from normal (N), isotype control antibody treated (IC) and neutrophil depleted (Gr-1) from day 11 pi mice, were analyzed by WB for VEGF-A (upper panel), sVR-1 (middle panel). Figure is a representative of two experiments and each experiment represent at least 3 corneas. (F) Relative fold change in the expression of VEGF-A and MMP-9 mRNA in neutrophil depleted and isotype antibody treated mice at day 11 pi as compared to naïve mock infected mice. Figure is a representative of two experiments and each experiment represent at least 4 corneas. Ly6G⁺ neutrophils were sorted from day 11 pi corneas. Cell suspension from 6–8 pooled corneas was used for neutrophil purification. Purified neutrophils were analyzed by either WB or RTPCR for the expression of VEGF-A. (G) Reducing WB analysis for VEGF-A from two different samples S1- Sample 1 and S2-Sample 2 shows the presence of VEGF-A protein from purified neutrophil samples. Data represent two different experiments and each sample represent neutrophils from 6–8 corneas. (H) Agarose gel analysis for β-actin (92bp) (lane 2) and VEGF-A (214bp) (lane 4) of cDNA from purified neutrophils. Lane 1 and 3 are reverse trancriptase negative control for β-actina and VEGF-A respectively. (I) Neutrophil depletion started from day 7 pi and continued until day 13 pi resulted in reduced angiogenesis as well as SK lesion severity. Data are representative of a two independent experiments (n = 8–10 mice/group). P = 0.0131 (*), P = 0.0068 (**).

Figure 7. Scheme illustrating the mechanism for HSV-1 mediated corneal angiogenesis

Normal uninfected cornea constitutively secretes large amount of sVR-1 and small amounts of VEGF-A. sVR-1 constrains the physiological effect of VEGF-A by binding it with very high affinity (Left panel). Early after corneal HSV-1 infection, there is sudden increase in the levels of VEGF-A primarily being produced by infected or nearby uninfected corneal epithelial cells. However, levels of sVR-1 go down mainly due to decreased production of sVR-1 by corneal epithelial cells and also due to degradation by MMP-2, MMP7 and MMP9. This in turns leads to more physiologically active VEGF-A, which is now free from inhibitory effect of sVR-1. This active form of VEGF-A drives the initial angiogenic sprouting early after HSV infection (middle panel). During chronic phase of HSK, when HSV-1 is no longer detected in the cornea, inflammatory cells particularly neutrophils act as a source of VEGF-A and different MMPs. This second wave of VEGF-A and MMPs further maintains the angiogenic response by continuous supply of active form of VEGF-A and MMPs, which degrades sVR-1. The development of new leaky blood vessels leads to the release of more plasma and inflammatory cells in the cornea thus setting the stage for chronic SK and blindness (Left Panel).