Corneal Hemangiogenesis After High-Risk Keratoplasty

Even after high-risk keratoplasty, preexisting blood vessels tend to increase. Only after keratoplasty for herpetic keratitis does removal of the angiogenic stimulus lead to a reduction in corneal angiogenesis. Animal experiments recently clearly demonstrated that even after high-risk keratoplasty there is a significant further increase in both hemand lymphangiogenesis. In addition, inhibition of these processes even after high-risk keratoplasty (in the mouse model) could improve subsequent graft survival. That is true both for “hot” eyes shortly after an inflammation and also for eyes where the inflammation has calmed down and vessels have partly regressed over months (“intermediate risk”). Even in these regressed old high-risk eyes does postkeratoplasty anti-VEGF therapy promote graft survival in the animal model.

Summary for the Clinician

•Corneal angiogenesis postoperatively occurs in about 50 % of patients after low-risk keratoplasty in preoperatively avascular recipient beds.

•Animal experiments suggest that hemangiogenesis after keratoplasty is accompanied by clinically invisible lymphangiogenesis.

•Postoperative hemand lymphangiogenesis have been identified as risk factors for immune rejection after keratoplasty.

•Inhibition of postkeratoplasty angiogenesis and lymphangiogenesis improves graft survival both in the low-risk and high-risk setting (mouse experiments)

•Care should be taken with contact lens-induced corneal angiogenesis in keratoconus patients, since that may compromise the success of a subsequent keratoplasty due to increased risk of immune rejections and may cause interface bleeding after DALK surgery.

Corneal Lymphangiogenesis: Essential for Corneal Graft Rejection

Lymphangiogenesis has recently gained wide interest for its important role in tumor metastasis and induction of alloimmunity after organ transplantation [21]. Whereas it has been known for more than 100 years that the normally avascular cornea can be invaded by blood vessels (hemangiogenesis), it was unclear until very recently whether the normally alymphatic human cornea can be invaded by lymphatic vessels from the lymphatic arcade at the limbus [1, 11]. The main reasons for that unclarity were (1) the fact that lymph vessels – in contrast to erythrocyte-filled blood vessels – are not detectable biomicroscopically using the normal slit-lamp magnification and (2) the lack of specific markers for lymphatic endothelium. The latter has changed in the last 5–10 years with the advent of several specific markers of lymphatic endothelium (such as LYVE-1, Podoplanin, and VEGF receptor 3 [21]). These novel markers enabled for the first time the precise identification of

lymphatic vessels in vascularized human corneas [11]. Lymphatic vessels were significantly more common in corneas with a short history of a corneal inflammation (usually keratitis or trauma) and also were significantly more common in heavily vascularized corneas [8]. Thereby, the chance of having both pathological blood as well as clinically invisible lymphatic vessels present is strongly correlated with the degree of corneal angiogenesis, which can be judged by slit-lamp evaluation. Furthermore, recent work shows that it is possible to demonstrate lymphatic vessels in vivo in the cornea using confocal microscopy (HRT II using the Rostock module; see below). Since lymphatic vessels are invisible at slit-lamp magnifications, they might not be as detrimental for corneal transparency as blood vessels are. In fact, animal experiments suggest that the “antilymphangiogenic privilege” of the cornea is not redundantly organized.

Using the mouse model of corneal neovascularization, we recently could demonstrate that after an inflammatory stimulus to the cornea, there is usually parallel and very early (within 48 h) outgrowth of both blood and lymphatic vessels. Both originate from the limbal vascular arcade [7]. The cornea therefore is also an excellent model system to study the mechanisms not only of angiogenesis but also lymphangiogenesis and test pharmacologic compounds for the relative inhibition of both processes in the animal model [14]. Compared to blood vessels, lymphatic vessels tend to regress much quicker and more complete after an inflammatory challenge to the cornea [22]. For example, after a short, 2-week long inflammatory stimulus (corneal sutures), all lymphatic vessels in the mouse cornea are completely regressed after 6 months, whereas blood vessels persist (partly as non-perfused ghost vessels) indefinitely. As outlined below, this supports the clinical practice not to perform penetrating keratoplasties in freshly inflamed eyes, but to wait until inflammation has calmed down to improve graft survival [22]. Lymphangiogenesis is mediated by the VEGF family growth factors VEGF-A, -C and -D as well as by FGF and PDGF [21]. Stimuli for the release of the main lymphangiogenic growth factor VEGF-C are primarily inflammatory in nature, explaining the clinical observation that human corneal lymphangiogenesis is more common shortly after keratitis [8].

The relative higher importance of the afferent lymphatic arm of the immune response in dictating the outcome of corneal graft survival has recently been demonstrated by several elegant studies: Indefinite survival of both fully mismatched orthotopic non-high-risk grafts [23] as well as 90 % survival of fully mismatched high-risk corneas [24] in BALB/c-mice was achieved by removal of cervical lymph nodes by cervical lymphadenectomy. Furthermore, pharmacologic strategies inhibiting lymphangiogenesis(and angiogenesis) after low-risk keratoplasty (see Fig. 5.2) and even after high-risk keratoplasty can significantly improve corneal graft survival. In addition, we were recently able to create differentially prevascularized recipient beds in mice. This allowed us to compare the survival rates of grafts placed into completely avascular recipient beds (low-risk setting) as compared to both hemand lymphvascularized recipient beds (high-risk setting) as compared to only hemvascularized (so-called alymphatic high risk) beds [10] (Fig. 5.2). Interestingly, that showed that there was no difference in survival rates between grafts placed into avascular as compared to grafts placed into only hemvascularized beds. Only if

lymphatic vessels were present in addition to blood vessels, the survival rate dropped significantly. That clearly shows that lymphatic vessels are the main “culprit” responsible for the high rate of immune rejections after keratplasty into high-risk recipient beds. All this supports the novel concept that antilymphangiogenic therapies can modulate immune responses after keratoplasty and thereby improve graft survival [10].

Fig. 5.2 Lymphatic vessels in the recipient bed prior to transplantation determine graft survival: Graft survival was significantly better when transplants were placed into recipient beds lacking lymphatic vessels (red line, green line, blue line) compared to beds with lymphatic vessels being present at the time of transplantation (black line) [10]. The presence or absence of blood vessels was not relevant (compare green [d: only blood vessels present] versus red line [c: no vessels at all]). Generation of different transplantation models. Images showing wholemounts of (c) avascular high-risk (inflamed, but avascular, recipients), (d, e) alymphatic high-risk (inflamed and hemvascularized, but no lymphatic vessels), (f) high-risk (inflamed and hemvascularized and lymphvascularized) and (g) normal risk (avascular) recipient beds as transplantation models

Fig. 5.2 (continued)

Summary for the Clinician

•During corneal inflammation, there is parallel outgrowth of both blood and lymphatic vessels from the limbus into the cornea (combined hemangiogenesis and lymphangiogenesis).

•Lymphatic vessels are more common in heavily vascularized human corneas and are more common shortly after a corneal inflammation (keratoplasty, keratitis, immune rejection, etc.)

•Corneal lymphatic vessels determine the fate of a graft placed into a highrisk cornea.

Corneal Lymphangiogenesis in Dry Eye

There is preliminary evidence suggesting an involvement of corneal lymphangiogenesis in the disease process of chronic inflammatory dry eye disease. Experimentally, in dry eye models, mild isolated ingrowths of lymphatic vessels into murine corneas can be observed. These lymphatics may act as conduit for the delivery of antigens to regional lymph nodes, thus initiating the chronic “autoimmune” form of dry eye disease. Novel anti(lymph)angiogenic treatment approaches may thus in the future also be part of dry eye treatment.