Abstract
Purpose.:
Lymphangiogenesis (LG) accompanies many corneal diseases after inflammatory, infectious, or chemical insults and is a primary mediator of transplant rejection. The purpose of this study was to investigate whether there is a time window for therapeutic intervention of corneal LG and whether a combined blockade of VEGFR-2 and VEGFR-3 effectively suppresses early-, middle-, or late-stage LG.
Methods.:
Corneal inflammatory neovascularization was induced by a standard suture placement model in mice. Neutralizing antibodies against VEGFR-3 and/or VEGFR-2 were administrated systemically with the treatment started at postoperative day 0, day 7, or day 14. Whole mount corneas were sampled for immunofluorescence microscopic studies using LYVE-1 (a lymphatic marker) antibodies. Digital images were analyzed by software.
Results.:
Both VEGFR-3 and VEGFR-2 were involved in corneal suture-induced inflammatory LG. Their combined blockade led to a significant inhibition of both early- and middle-stage LG while demonstrating no effect on late-stage LG.
Conclusions.:
Corneal inflammatory LG has a discrete time window for intervention therapy. Although it is important to start the treatment as soon as possible, interventions initiated in the middle of the LG process are still effective. These novel findings will shed some light on our understanding of inflammatory LG and the development of new therapeutic protocols for LG-related diseases at different stages.
Lymphatic research has experienced exponential growth in recent years largely because of the advancement of technologies and the discoveries of several lymphatic endothelial specific molecules, including lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1), Prox-1, and VEGFR-3. The lymphatic network penetrates most tissues in the body, and its dysfunction has been found in a broad spectrum of disorders, such as cancer metastasis, inflammation, transplant rejection, obesity, and hypertension.
1 –6 To date, there is still little effective treatment for lymphatic diseases; it is, therefore, a field with an urgent demand for new therapeutic protocols.
Given its accessible location and transparent nature, the cornea provides an optimal site for lymphatic research. Although the normal cornea does not have lymphatic vessels, lymphangiogenesis (LG; the development of new lymphatic vessels) can be induced in this tissue after inflammatory, traumatic, chemical, or infectious damage.
7 –9 LG also constitutes the afferent arm of the immune reflex arc of corneal transplantation immunity,
7,9,10 and, most recently, it has been demonstrated that LG is a primary mediator of corneal transplant rejection.
11
The VEGF family is the backbone of a complex network controlling blood and lymphatic vessel processes. A number of previous studies have shown that VEGFR-3 mediates LG in the cornea and other tissues, and its inhibition suppresses transplant rejection, tumor growth, and metastasis.
11 –17 The specific role of VEGFR-2 in LG, however, has been much less studied. It is known that this factor is critically involved in hemangiogenesis (HG; the development of new blood vessels) and that its inhibition suppresses HG and tumor growth.
18 –21 Most recently, it has been indicated that VEGFR-2 also plays a role in corneal LG,
12,22,23 though the underlying mechanisms still remain largely unknown.
Although results from previous corneal studies on LG are promising, there is still a large knowledge gap when translating these results to clinic settings. For example, almost all the previous studies focused on how to prevent LG by testing an intervention started right before, or on the same day as, a pathologic stimulation, which happens only infrequently in patients. Indeed, many patients who need LG treatment miss their first chance to receive emergent care, possibly because of difficult living conditions, transportation problems, or insufficient knowledge about the disease process itself. Indeed, LG is a progressive event that can occur insidiously at its early phases.
In this study, we first demonstrate that VEGFR-2 is involved in suture-induced inflammatory LG but to a lesser extent than VEGFR-3. More important, we reveal that there is a discrete time window for therapeutic intervention of corneal inflammatory LG. We show that a combined blockade of VEGFR-2 and VEGFR-3 suppresses both early- and middle-stage LG while demonstrating no effect on late-stage LG. Close correlation between the timing and outcomes of the treatment is also defined. It is hoped that our results will provide a useful guide for understanding of the LG processes and the development of novel therapeutic protocols for LG-related diseases at various stages.
Six- to 8-week-old male BALB/c mice (Taconic Farms, Germantown, NY) were used for these experiments. All mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and all protocols were approved by the Animal Care and Use Committee of the University of California at Berkeley. Mice were anesthetized using a mixture of ketamine, xylazine, and acepromazine (50 mg, 10 mg, and 1 mg/kg body weight, respectively) for each surgical procedure.
In this study, we provide the first evidence showing that corneal inflammatory LG has a discrete time window for intervention therapy. At least two important conclusions can be drawn from our data: combined blockade of VEGFR-2 and VEGFR-3 is effective in suppressing corneal LG at its early and middle stages; though it is important to initiate treatment as soon as possible, any interventions falling within the therapeutic time window should still be effective. However, a significant time delay will result in a loss of treatment efficacy.
VEGFR-2 is an emerging factor for LG research.
12,22,23 Our data derived from the suture placement model are consistent with recent findings showing that corneal LG induced by VEGF-C or VEGF-D pellets is regulated by VEGFR-2.
12,22 Given that the doses of the growth factors used in the micropocket assays are much higher than physiological levels, results from the present study should more closely mimic patients' conditions and thus provide additional data supporting VEGFR-2 interference as an adjunctive treatment for LG. Additionally, because the VEGFR-2 pathway also plays a major role in HG,
12,18 –21 a process accompanies LG and contributes to tissue injury after myriad corneal insults, combined blockade of VEGFR-2 and another key lymphatic receptor, such as VEGFR-3, will maximize the treatment effect when significant inhibition on both processes is pursued.
Moreover, this study also provides a new guideline for modulating corneal LG. Because LG is a progressive process, it is imperative to determine appropriate strategies for intervention therapy. Our finding that the combined blockade strategy is almost equally effective in suppressing early- and middle-stage LG bears broad implications. Most important, it will help to define a suitable population for treatment, which is larger than previously assumed. As indicated by this study, this population should include patients not only immediately after corneal damage but also those who have missed the chance for emergent care and are still in the middle of LG processes. Although it is yet to be determined how the combined treatment suppresses middle-stage LG, it is plausible to hypothesize that the outcome is a net effect of a prevention of LG progression, a disturbance of LG maturation or induction of LG regression, or both. Further analysis of this exciting finding may, therefore, yield crucial insight into the mechanisms of various aspects of LG.
Last, LG is a primary mediator of corneal transplant rejection. In the high-risk setting in which grafting is performed in the inflamed and lymphatic-rich beds, the rejection rate can be as high as 90%.
7,9,10 At this stage, there is little effective treatment. Unfortunately, many patients who are in need of corneal transplantation fall into this category. Our study indicates that we may be able to modify or normalize the high-risk grafting beds, particularly when they are still at the early or middle stages of LG, by the combination strategy to improve the survival rate of high-risk transplants; this warrants further investigation.
Supported in part by research grants from the National Institutes of Health, the Department of Defense, and the University of California at Berkeley (LC).
Disclosure:
D. Yuen, None;
B. Pytowski, ImClone Systems (E);
L. Chen, None
The authors thank Jeffrey LeDue and Steven Ruzin (University of California at Berkeley) for their excellent technical assistance with the immunofluorescence microscopic studies.