Novel Characterization of Lymphatic Valve Formation during Corneal Inflammation

Lymphatic research has progressed rapidly in recent years. Though lymphatic dysfunction has been found in a wide array of disorders from transplant rejection to cancer metastasis, to date, there is still little effective treatment for lymphatic diseases. The cornea offers an optimal site for lymphatic research due to its accessible location, transparent nature, and lymphatic-free but inducible features. However, it still remains unknown whether lymphatic valves exist in newly formed lymphatic vessels in the cornea, and how this relates to an inflammatory response. In this study, we provide the first evidence showing that lymphatic valves were formed in mouse cornea during suture-induced inflammation with the up-regulation of integrin alpha 9. The number of corneal valves increased with the progression of inflammatory lymphangiogenesis. Moreover, we have detected lymphatic valves at various developmental stages, from incomplete to more developed ones. In addition to defining the average diameter of lymphatic vessels equipped with lymphatic valves, we also report that lymphatic valves were more often located near the branching points. Taken together, these novel findings not only provide new insights into corneal lymphatic formation and maturation, but also identify a new model for future investigation on lymphatic valve formation and possibly therapeutic intervention

sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo

We would like to thank Jonathan Winger and Xiao Zhu for guidance with the insect cell protein expression system and providing reagents. We would like to acknowledge Ann Fischer for help with expressing the sFlt protein in the Tissue Culture Facility at UC Berkeley and Dawn Spelke and Anusuya Ramasubramanian for help optimizing protein purification from insect cells. We are also grateful for the help from Leah Byrne and John Flannery at in the Helen Wills Neuroscience Institute at UC Berkeley for aiding us in the development of the rat intravitreal residence time model and for allowing us to use their facilities.Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.Yeshttp://www.plosone.org/static/editorial#pee

Improving Anti-VEGF Drugs in the Vitreous

The work described in this dissertation present a novel technique utilizing multivalent hyaluronic acid bioconjugates with an anti-VEGF protein for improving the action of drugs in the vitreous. This technology, which has shown efficacy both in vitro and in vivo has the potential to enhance the bioactivity of drugs used for treating patients with diseases including diabetic retinopathy, wet AMD and other neovascular diseases of the retina.Chapter 2 described our initial efforts in creating multivalent conjugates of the anti-VEGF protein, sFlt. Before beginning in vivo studies, we wanted to determine what parameters would maximize the bioactivity of mvsFlt. We investigated the use of several HyA molecular weights and valencies of sFlt molecules to HyA chains. The characterization and in vitro experiments were carried out with 6 mvsFlt conjugates of 300 kDa, 650 kDa and 1 MDa molecular weights with feed ratios of 10 sFlt per 1 HyA chain (termed low conjugation ratio (LCR)) and 30 sFlt per 1 HyA chain (termed high conjugation ratio (HCR)). SDS-PAGE and SEC-MALS enabled us to examine the conjugation efficiency following the reaction as well as to investigate the composition of the conjugates focusing specifically on the contribution of unbound sFlt that remained in solution. The in vitro experiments were crucial in determining whether the conjugation of sFlt to the HyA resulted in a decrease in the affinity of sFlt for VEGF. Using an ELISA and a cell-based survival assay, we determined that all the conjugates, irrespective of their molecular weight and valency, equally inhibited VEGF activity and were unaffected by conjugation. Using HyA crosslinked gels, we created an in vitro model of the vitreous to study how the increase in size impacted movement of the mvsFlt conjugates through the gel. The largest mvsFlt conjugates (650 kDa and 1 MDa) were significantly slower in their movement through the gel. The 650 kDa mvsFlt conjugate was then used in the in vivo studies described in Chapter 3. Furthermore, this conjugate was used to confirm the protective effect of HyA on sFlt degradation by a protease that specifically targets sFlt, matrix metalloproteinase-7. Taken together, the work in this chapter demonstrated the efficacy of conjugation, characterization, in vitro bioactivity, slowed diffusion and protection from proteases. The progress demonstrated in this chapter enabled the studies presented in Chapter 3. The work in Chapter 3 details the in vivo studies used to show mvsFlt efficacy in two models of in vivo angiogenesis and a half-life model in the rat vitreous. As shown in Chapter 2, all the mvsFlt conjugates performed equally at inhibiting VEGF-dependent processes. Thus, we chose the conjugate that displayed slowest diffusion in the crosslinked HyA vitreous model, the 650 kDa mvsFlt conjugate. The first question we tried to address was whether the mvsFlt conjugate remained bioactive in vivo. The corneal angiogenesis model enabled us to examine the effect of sFlt and mvsFlt treatment on the growth of blood vessels in a corneal injury model of angiogenesis. Due to the fact that VEGF is one of the main mediators of angiogenesis in vivo, we expected that the addition of sFlt and mvsFlt would inhibit the formation of new blood vessels. Our data showed that both the sFlt and mvsFlt were equally capable of inhibiting angiogenesis in this model, indicating that the conjugation did not decrease the ability of sFlt to inhibit angiogenesis in vivo. We next demonstrated that the conjugation of sFlt to HyA significantly enhanced the half-life of sFlt in the vitreous of the rat eye by an order of magnitude. Finally, we utilized an oxygen-induced retinopathy rat model to examine the indirect effect of increased half-life on the prolonged anti-angiogenic effect of mvsFlt on neovascularization in the retina. mvsFlt was more effective than sFlt at inhibiting retinal neovascularization, likely due to its increased half-life in the vitreous. Taken together, the mvsFlt conjugate showed superior activity to sFlt in vivo, results that could substantially improve upon currently available drugs for treating retinal disorders. Chapter 4 gives an overall conclusion and details future directions the project can go in. There are some questions that remain unanswered and this chapter is a guideline to help fill in those gaps

Improving Anti-VEGF Drugs in the Vitreous

The work described in this dissertation present a novel technique utilizing multivalent hyaluronic acid bioconjugates with an anti-VEGF protein for improving the action of drugs in the vitreous. This technology, which has shown efficacy both in vitro and in vivo has the potential to enhance the bioactivity of drugs used for treating patients with diseases including diabetic retinopathy, wet AMD and other neovascular diseases of the retina.Chapter 2 described our initial efforts in creating multivalent conjugates of the anti-VEGF protein, sFlt. Before beginning in vivo studies, we wanted to determine what parameters would maximize the bioactivity of mvsFlt. We investigated the use of several HyA molecular weights and valencies of sFlt molecules to HyA chains. The characterization and in vitro experiments were carried out with 6 mvsFlt conjugates of 300 kDa, 650 kDa and 1 MDa molecular weights with feed ratios of 10 sFlt per 1 HyA chain (termed low conjugation ratio (LCR)) and 30 sFlt per 1 HyA chain (termed high conjugation ratio (HCR)). SDS-PAGE and SEC-MALS enabled us to examine the conjugation efficiency following the reaction as well as to investigate the composition of the conjugates focusing specifically on the contribution of unbound sFlt that remained in solution. The in vitro experiments were crucial in determining whether the conjugation of sFlt to the HyA resulted in a decrease in the affinity of sFlt for VEGF. Using an ELISA and a cell-based survival assay, we determined that all the conjugates, irrespective of their molecular weight and valency, equally inhibited VEGF activity and were unaffected by conjugation. Using HyA crosslinked gels, we created an in vitro model of the vitreous to study how the increase in size impacted movement of the mvsFlt conjugates through the gel. The largest mvsFlt conjugates (650 kDa and 1 MDa) were significantly slower in their movement through the gel. The 650 kDa mvsFlt conjugate was then used in the in vivo studies described in Chapter 3. Furthermore, this conjugate was used to confirm the protective effect of HyA on sFlt degradation by a protease that specifically targets sFlt, matrix metalloproteinase-7. Taken together, the work in this chapter demonstrated the efficacy of conjugation, characterization, in vitro bioactivity, slowed diffusion and protection from proteases. The progress demonstrated in this chapter enabled the studies presented in Chapter 3. The work in Chapter 3 details the in vivo studies used to show mvsFlt efficacy in two models of in vivo angiogenesis and a half-life model in the rat vitreous. As shown in Chapter 2, all the mvsFlt conjugates performed equally at inhibiting VEGF-dependent processes. Thus, we chose the conjugate that displayed slowest diffusion in the crosslinked HyA vitreous model, the 650 kDa mvsFlt conjugate. The first question we tried to address was whether the mvsFlt conjugate remained bioactive in vivo. The corneal angiogenesis model enabled us to examine the effect of sFlt and mvsFlt treatment on the growth of blood vessels in a corneal injury model of angiogenesis. Due to the fact that VEGF is one of the main mediators of angiogenesis in vivo, we expected that the addition of sFlt and mvsFlt would inhibit the formation of new blood vessels. Our data showed that both the sFlt and mvsFlt were equally capable of inhibiting angiogenesis in this model, indicating that the conjugation did not decrease the ability of sFlt to inhibit angiogenesis in vivo. We next demonstrated that the conjugation of sFlt to HyA significantly enhanced the half-life of sFlt in the vitreous of the rat eye by an order of magnitude. Finally, we utilized an oxygen-induced retinopathy rat model to examine the indirect effect of increased half-life on the prolonged anti-angiogenic effect of mvsFlt on neovascularization in the retina. mvsFlt was more effective than sFlt at inhibiting retinal neovascularization, likely due to its increased half-life in the vitreous. Taken together, the mvsFlt conjugate showed superior activity to sFlt in vivo, results that could substantially improve upon currently available drugs for treating retinal disorders. Chapter 4 gives an overall conclusion and details future directions the project can go in. There are some questions that remain unanswered and this chapter is a guideline to help fill in those gaps

Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells

Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity

Morphologically distinct stages of lymphatic valve formation in inflamed cornea.

<p>Representative micrographs demonstrating a mature lymphatic valve in normal conjunctiva (<b>A</b>) and various stages of lymphatic valves in inflamed corneas 2 weeks after suture placement (<b>B</b>–<b>D</b>). (<b>B</b>) Spotted expression of Itga-9 at early stage of valve formation. (<b>C</b>) Thin ring of Itga-9 expression characteristic of intermediate stage of valve formation. (<b>D</b>) Late stage of valve formation identified by strong band of Itga-9 expression, similarly as seen in normal conjunctiva (<b>A</b>). Itga-9: red; LYVE-1: green. Original magnification: 400 X. Arrows: lymphatic valves.</p

Localization of corneal lymphatic valves at branching points.

<p>(<b>A</b>–<b>C</b>) Representative micrographs demonstrating the location of newly formed lymphatic valves at vessel branching points, as indicated by the arrows. Itga-9: red; LYVE-1: green. (<b>B</b> and <b>C</b>) Higher magnification views of the boxed areas in (<b>A</b>). Original magnification: 200 X (<b>A</b>) and 400 X (<b>B</b> and <b>C</b>).</p

Itga-9 expression is increased in inflamed cornea.

<p>(<b>A</b>) Representative micrographs from semi-quantitative RT-PCR analysis showing that Itga-9 expression is significantly increased in 2 week post-sutured corneas compared with normal control. (<b>B</b>) Summarized data from 3 repetitive experiments. ***<i>P</i><0.001.</p