Aim 2: To examine and compare adipogenic, lipogenic, lipolytic, immune, fibrotic and angiogenic parameters of healthy and lymphedema associated SAT in women.
Despite the fact that later stages of secondary lymphedema are associated with massive accumulation of AT, it is still rather obscure what the characteristics of this aberrant tissue are. Similarly, AT depots in the upper limbs belong to an understudied area of research compared to GAT.
Therefore, our study aimed to provide the first comprehensive analysis of lymphedema and non-lymphedema AT of the upper limbs. Adipocyte-related LAT qualities were compared between lymphedema affected and healthy paired limbs of lymphedema patients and healthy subjects. In addition, immune, fibrotic, and (lymph)angiogenic parameters of the LAT were also examined and compared. The results of our study showed that LAT-derived adipocytes are not hypertrophic compared with healthy adipocytes. These results are inconsistent with the data from the Prox1 ± mouse model [212] and the results of human studies analyzing adipocyte size in LAT in the lower and upper lymphedematous limbs, which show a hypertrophic pattern of adipocytes [185, 258]. Indeed, large adipocytes could be more fragile, so their isolation by collagenase digestion could artificially exclude them from the analysis. Although we are quite confident in our collagenase digestion protocol, as we have used it successfully in our other clinical studies, to resolve this bias, the size distribution patterns should be assessed by different approaches side by side (i.e., histology sections vs.
osmium tetroxide staining vs. collagenase digestion) when both the comparison between adipocytes from LAT and AT from 'healthy' limbs from lymphedema patients as well as AT from breast cancer survivors free of lymphedema should be performed. This comparison , however, may run into ethical and practical limits, as LAT is usually obtained by liposuction, making it impossible to obtain histologically quality specimens; moreover, it can be assumed that the willingness of cancer survivors to undergo a fat biopsy will be rather low.
In the animal model of secondary lymphedema, enhanced activity of PPARγ, which is the master regulator of adipogenesis and lipogenesis in adipocytes, has been detected in the subcutaneous area. Therefore, expansion of LAT could be driven through enhanced adipogenesis or lipogenesis. Nevertheless, expression pattern of analysed lipogenic markers was not substantially changed in diseased limb of LYM subjects compared to healthy, while
113 the expression of both inhibitors (GOT2,WISP2, INHBA) and activators (ZFP423, KLF9) of adipogenesis was enhanced. Accordingly, previous mRNA profiling in AT by microarrays did not detect any difference in the expression of typical lipogenesis or adipogenesis markers in healthy compared to diseased limb of lymphedema [309]. Interestingly, AT expression of only 40 genes (out of 16 000 genes) was found to be modified by lymphedema [309]. Thus, posttranscriptional regulation is probably more involved in the maintenance of the diseased phenotype of lymphedema.
In vitro, cells isolated from LAT differed from healthy cells by the increased expression of genes involved in the regulation of adipogenesis (both pro- and anti-adipogenic regulators) that however did not result in the increased ability to undergo adipogenesis in vitro. In fact, KLF4 and KLF6, expression of which was correlated, are transcription factors with the mixed role in the regulation of adipogenesis [310]. Furthermore, we did not prove higher proliferative activity of LAT preadipocytes in vitro. Nevertheless, we have to admit that the donors of LAT cells were older and fattier compared to healthy donors and all these factors could negatively affect the adipogenic and proliferative capacity of cells (as shown before by us and others [286, 311] and thus possible obscure differences between the cells.
Not only altered adipogenesis and lipogenesis but also decreased lipolysis could contribute to AT expansion seen in secondary lymphedema. However, basal lipolysis in LAT seems to be rather increased, not decreased, as documented by higher glycerol and FFAs levels released ex vivo from AT explants derived from diseased limbs. In addition, measurement of the expression levels of lipolytic and lipid handling genes was performed, which confirmed the previously described increased levels of lipolytic products in the affected limb. PEDF was shown to be a potent activator of ATGL, one of the major AT lipases [122].
Higher basal lipolysis in the LAT correlated with higher PEDF and glycerol levels in media conditioned with ex vivo AT explants. PLIN1 and PLIN3 expression was increased in the LAT, along with a modest increase in mRNA levels of HSL, another important AT lipase.
Recent study, provided by Luo et. al. [118] has shown that PEDF play active role as new lymphangiogenic inhibitor described in nude mouse model. Otherwise, the most interesting finding was the higher expression of the PDPN, one of LVs markers [179, 312], in diseased limb of AT compared to the AT of healthy limb and healthy subjects. The correlation of ATGL/PLIN mRNA levels with PDPN/PEDF mRNA levels together with basal glycerol release supports a newly suggested link between lipolysis and lymph-angiogenesis.
114 Enhanced fibrosis in human LAT has been shown previously using histological methods [185] and by mRNA profiling in AT [309]. We were able to confirm the higher mRNA levels of TNC found by Soupe et al. [313] TNC was found to promote the formation of poorly functional blood vessels via regulation of Wnt signaling. Interestingly, TNC was found to be elevated in visceral (but not subcutaneous) AT in obese humans [314], i.e., in the AT depot with an intimate connection with intestinal lymphatics.
Otherwise, as described previously, inflammation is a critical component of the pathophysiology of secondary lymphedema [254]. Therefore, it is possible that AT deposition in lymphedema and altered metabolic function could be related not only to different mechanisms regulating adipogenesis, lipolysis, and (lymph)angiogenesis, but also to greater inflammatory reactivity. Murine model of lymphedema revealed a substantial increase in the numbers of CD4+ T lymphocytes and macrophages upon surgical damage to LS [271]. The increased infiltration of lymphedematous tissue by CD4+ T lymphocytes persisted for 6 weeks after the axillary lymph node dissection, while increased numbers of macrophages were evident for only 3 weeks but not in later stages. Similarly, in our study analyzing samples from chronic stages of secondary lymphedema, we found a higher relative content of activated CD4+ T lymphocytes but no significant increase in the content of macrophages when compared to healthy AT. The lack of higher infiltration of LAT by macrophages is also in line with the findings of the study by Tashiro et al. [185], probably the only available human study describing immune cell content in LAT, that reported an even lower number of CD45+ hematopoietic cells and crown-like structures in lower extremity LAT compared to AT from the healthy limb. Concerning higher content of CD4+
T lymphocytes, it is however questionable whether the overall signalling originating from CD4+ T lymphocytes is substantially altered as the balance between the individual subtypes of CD4+ T lymphocytes was similar in both types of AT. Besides ICs, the expression of pro- inflammatory cytokines is another sign of tissue inflammation. In this case, we have proved much higher secretion of 5 pro-inflammatory cytokines from ex vivo cultivated LAT explants. The strikingly increased secretion of these pro-inflammatory cytokines was however in contrast with their almost unchanged mRNA levels – in fact, mRNA level of MCP1 was actually higher in healthy limb compared to diseased limb in LYM subjects and the similar trend was observed for IL8. No difference in mRNA levels of IL6 and IL8 between healthy and diseased limb AT of lymphedema patients was also reported in the first mRNA profiling study of AT from healthy and diseased limbs of lymphedema patients
115 [309]. Thus, higher secretion of pro-inflammatory cytokines from LAT may be a result of either strongly enhanced stability or translation of these mRNAs or mechanical release of cytokines from the interstitial space of AT, where they may be 'trapped'. A later hypothesis could also be supported by the fact that no difference was found in the plasma levels of these cytokines that are partially dependent on the production from AT.
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