The presence of newly formed lymphatic vessels in the alveolar spaces of IPF lung suggests a role for soluble lymphangiogenic factors in the alveolar milieu.30
Bronchoalveolar lavage fluid (BALF) from subjects with IPF triggered significantly more migration of lymphatic endothelial cells (LEC) than did healthy volunteer BALF.
Surprisingly, VEGF-C and VEGF-D, known to play key roles in lymphangiogenesis in disease,37
did not seem to be driving lymphangiogenesis in IPF. VEGF-C levels were lower in IPF BALF than in that from healthy volunteers; levels of VEGF-D were similar.30
VEGF-A, an angiogenic growth factor, is known to induce lymphangiogenesis in inflammatory conditions. In mice overexpressing VEGF-A in the epidermis, triggering a delayed hypersensitivity reaction resulted in increased lymphangiogenesis, which can be significantly attenuated with function-blocking antibodies against VEGFR-1 and VEGFR-2.38
Moreover, in inflammation, VEGF-A can trigger lymphangiogenesis indirectly, via recruitment of macrophages, which in turn secrete VEGF-C and VEGF-D.39
In line with other studies,17,40,41
we found the levels of VEGF-A in IPF BALF to be markedly decreased, strongly suggesting the absence of a significant role for VEGF-A in the lymphangiogenic process.
Levels of fibroblast growth factor, which is known to induce lymphangiogenesis,42
and TIMP-2, which stabilizes vascular tubes,43
were not elevated in BALF of IPF subjects. Conversely, the levels of hepatocyte growth factor, which induces lymphangiogenesis during tissue repair and inflammation44
were higher in IPF BALF.30,45
In addition, levels of TIMP-1 and CCL-246,47
were higher in IPF BALF. Addition of function-blocking antibodies against HGF, TIMP-1, and CCL2 to IPF BALF did not change migration of LEC.30
Hyaluronan (HA) is known to play a key role in lung injury and repair,48
and to induce angiogenesis in a bleomycin-induced lung injury model.49
More recently, the role of HA in tumor lymphangiogenesis has been examined.50,51
In a study of tumor lymphangiogenesis, exogenous HA induced intratumoral lymphangiogenesis, but not angiogenesis.50
Mammary tumor cells overexpressing hyaluronan synthase 2 significantly increased intratumoral lymphangiogenesis in parallel with the development of stromal tissue.51
Stromal cells, particularly tumor-associated fibroblasts, seem to play a key role in the lymphangiogenic process by upregulation of VEGF-C and -D production. A direct role for HA in lymphangiogenesis could not be excluded.51
In our study,30
short-fragment HA in IPF BALF increased LEC migration and proliferation. The addition of short-fragment HA to healthy volunteer BALF increased LEC migration. These data suggest a direct role for hyaluronan in lymphangiogenesis in IPF. There are multiple known receptors for HA on the surface of blood endothelial cells.52,53
Savani et al.53
have shown that migration and proliferation are induced by HA receptors CD44 and CD168, respectively. These receptors seem to be redundant,54
since in CD44 null-mice, CD168 more efficiently sustained collagen-induced inflammation.
In inflammation and cancer, new lymphatic vessels arise from existing ones. Our data and those of others23,30
showed that newly formed alveolar lymphatic vessels were not connected to existing lymphatics, consistent with a role for a lymphatic progenitor endothelial cell.55
There is, however, conflicting evidence about the contribution of progenitor cells to lymphangiogenesis. Salven et al.56
described a CD34+
circulating progenitor endothelial cell, with the potential to differentiate into mature blood vascular and lymphatic endothelial cells in vitro
. In cancer and metastasis, preexisting lymphatic endothelium appears to play a critical role in lymphangiogenesis, with little or no role for a bone marrow-derived endothelial progenitor cell.57
In humans, circulating progenitor lymphatic endothelial cells were first described in sex-mismatched kidney transplant recipients. After transplant rejection, lymphatic endothelial cells from the recipient were found in the donor kidney.55
A recent report58
showed that human mesenchymal stem cells grown in conditioned medium of human microvascular endothelial cells are capable of differentiating to LEC in vitro
, and in vivo
can restore lymphatic function in a mouse model of lymphedema.
Maruyama et al.59
were the first to show that activated CD11b+
macrophages were able to differentiate into LEC both in vitro
and in vivo
in a corneal inflammation model. Earlier findings demonstrated that CD11b+
macrophages are increased in IPF BALF.60
In our study, CD45+
macrophages were isolated from BALF of subjects with IPF and healthy volunteers, and subsequently grown in Matrigel®
. No quantitative difference in CD11b+
macrophages was detected, but functional differences between CD11b+
macrophages isolated from IPF BALF and healthy volunteers were observed. CD11b+
macrophages, isolated from IPF BALF and grown in Matrigel, formed tube-like structures (), whereas CD11b+
macrophages isolated from healthy volunteers did not. These tube-like structures expressed LEC markers LYVE-1 and podoplanin.
FIG. 2. CD11b+ alveolar macrophages in IPF develop tube-like structures in vitro. CD11b+ alveolar macrophages were cultured in Matrigel for up to 31 days and inspected under white light, or after fluorescent labeling of cytoplasm (CellTracker Orange CMTMR) and (more ...)
The role(s) of these newly formed lymphatic vessels in IPF remains to be investigated. We hypothesize () that they may contribute to the fibrotic process and the maintenance of the injury. Several mechanisms may play a role in this process: first, LEC produced CCL21 a chemokine,61
which was elevated in IPF BALF.30
CCL21 induced proliferation of IPF-derived fibroblasts via stimulation of its receptor CCR-7.62
Moreover, in IPF, CCL21 stimulated recruitment of dendritic cells,63
which might contribute to the persistence of the injury. Finally, in a different model of tissue fibrosis,64
inhibition of transforming growth factor (TGF)-ß1 resulted in acceleration of lymphatic regeneration, raising the possibility that the fibrotic process itself negatively regulates lymphangiogenesis.
FIG. 3. Lymphangiogenesis in IPF: mechanisms and potential role. The deposition of hyaluronan after lung injury leads to lymphatic endothelial cell (LEC) migration and proliferation. Lung injury leads to the activation of CD11b+ macrophages, which can transdifferentiate (more ...)
In summary, lymphangiogenesis is part of the disease process in IPF. Hyaluronan and CD11b+ macrophages seem to drive, at least partially, lymphangiogenesis in IPF. The role(s) of these lymphatic vessels and the interplay between fibrosis and lymphangiogenesis remain to be investigated. Understanding pathways leading to lymphangiogenesis may offer novel therapeutic targets in IPF.