Lymphangioleiomyoma Cells and Lymphatic Endothelial Cells

Abstract

This Correspondence addresses the use of LAM and LAM-derived cells in Issaka et al. (Am J Pathol 2009,175: 1410–1420)

To the Editor-in-Chief:

We have read the article by Issaka et al1 with great interest as well as deep concern. They reported that lymphangioleiomyoma (LAM) cells express vascular endothelial growth factor (VEGF) receptor (R)-3 and, in turn, that autocrine and paracrine signaling via the VEGF-C/VEGF-D/VEGFR-3 axis plays a role in LAM cell biology in vivo. The source of their LAM-derived cells (LDCs)2,3 was a clone established from LAM nodules in patients’ lungs.

We commend the authors’ enthusiasm for their results, but have found several inaccuracies. First, although they admit that the LDCs they tested were not LAM cells in vivo, they state in the text as well as the article’s title that LAM cells in vivo grow through autocrine cross talk with lymphatic endothelial cells (LECs). Second, they kindly cite our studies reporting that LAM tissues consist of abundant VEGFR-3-positive LECs and that LAM is characterized by an association with lymphangiogenesis.4,5 VEGFR-3 is one of the most well-known markers of LECs.6 Although we provided concrete evidence that our VEGFR-3-positive cells were LECs, we have never stated that LAM cells are VEGFR-3 positive.4,5

In Figure 4 of their study, Issaka et al state that LAM cells are positive for VEGFR-3. However, the lung tissues illustrated in Figure 4 do not have VEGFR-3-positive-LECs. Despite the fact that many LAM cells stained positive for VEGFR-3 in this figure, no LECs stained positively for VEGFR-3. We have documented immunostaining of LAM tissue with anti-VEGFR-3 numerous times4,5,7 but have never obtained results like those reported by Issaka et al. Our immunostaining in LAM tissues in vivo using the same methods as described previously demonstrates that LECs are positive for VEGFR-3, whereas LAM cells are negative.4,5,7 Double immunostaining with α-smooth muscle actin (SMA) as a marker of LAM cells and VEGFR-3 as a marker of LEC as well as immunofluorescence using SMA and VEGFR-3 clearly indicate a separation between SMA positive and VEGFR-3 positive cells, with little overlap (unpublished data).

Therefore, we question their use of prox-1 immunostaining to show the existence of LECs in LAM tissue and their claim that perilymphatically-located LAM cells were positive for VEGFR-3. In our hands, double immunostaining for prox-1, a transcription factor specific for LEC, and VEGFR-3 revealed VEGFR-3-positive cytoplasm with prox-1-positive nuclei in only a subset of VEGFR-3 cells (unpublished data). These data suggest that not all LECs are transcriptionally active; that is, some LECs are VEGFR-3-positive but prox-1-negative, whereas some are both VEGFR-3- and prox-1-positive.

When LAM cell clusters (LCCs), isolated from chylous effusion, are cultured, they break into LAM cells and LECs in 5 to 7 days.5 Using this model, polygonally shaped LECs are positive for VEGFR-3, whereas spindle-shaped and SMA-positive LAM cells are negative for VEGFR-3 (unpublished data). Importantly, LCCs are genuine LAM cells5,7 and differ from LDCs, where clones are isolated from lung tissue after multiple procedural steps and 3 to 10 rounds of passages.1 Indeed, LCC-derived cells are maintained in vitro for a very short period and are thus are highly likely to retain the physiological properties of their status in vivo, in contrast to the cloned LDCs that Issaka et al used throughout their experiments.1 If the results of the immunohistochemical analysis of LAM tissues (Figure 4, A and B) by Issaka et al were truly correct, we would have to admit that a great difference must exist in the pathogenesis of LAM between the patient populations examined in our studies.

It is now widely accepted that LAM is a neoplastic disease and progresses through metastasis, although LAM cells appear histopathologically benign. However, LAM is a “chronic and slowly progressive neoplasm,” leading to destruction of the lungs in a course that is quite distinct from, for example, that of lung cancer, a representative malignant neoplasm of the lungs. Patients with LAM have about a 70% survival rate at 10 years after diagnosis,8,9 which is much longer than that of individuals with lung cancers. Accordingly, LAM patients are candidates for lung transplantation, although LAM can recur in the donor lung. This point seems to have been missed in research on LAM cell biology. LAM cells do not proliferate in vivo as fast as in vitro; therefore, the gap between results gained under circumstances most closely duplicating fresh living cells versus those in longer-term culture would be much bigger for LAM cell biology than for cancer cells.

In addition, the precise identity of LAM cells remains unclear.10 LAM tissues are composed of heterogeneous cells with different morphologies and immunoreactivities.10 Accordingly, the differing appearances of cells in LAM lesions are associated with cell-specific functions, which may help to explain why LAM patients have variable clinical courses.10 Conceivably, the clones Issaka et al used may be just one phenotype of LAM cells consisting of LAM lung tissues. However, that type must be a small minority of the overall content in LAM lesions judging from our consecutive results of repeated immunohistochemical analyses.4,5,7 From the viewpoint of physician-scientists caring for LAM patients, we believe that LAM researchers must bear in mind whether the choice of cells used in experiments and the role those cells play in vivo are truly relevant for human application.