Our results with SV40- lacZ mice indicate that in the normal bladder, a rich lymphatic vessel network is visible from the adventitia through the detrusor smooth muscle. It is characterized as a vascular network of blind ended, thin-walled capillaries that merge to larger collecting ducts, all positively stained with LYVE-1 antibody. In sharp contrast with the dense blood vessel vascularization of the bladder mucosa, we found that lymphatic vessels are absent of the sub-urothelium being located much deeper in the lamina propria (Figures 6A, 6B, 7A, and 7B) which seems to parallel the clinical findings in the human bladder [35, 36].
Our analysis indicates an increased lymphatic density during cancer progression and a co-localization of Ki-67 with some of LYVE-1-positive lymphatics, suggesting cancer-induced lymphangiogenesis (Figure 4), as it was recently suggested in human bladder cancer [10, 37]. However, the mechanisms for bladder lymphangiogenesis are not clear. In contrast to blood vessel angiogenesis, the mechanisms of lymphangiogenesis in general are still relatively vague . Vascular endothelial growth factor-C (VEGF-C) and VEGF-D have been implicated as specific regulators of lymphangiogenesis [39–43]. Both growth factors mediate their biological activity mainly by VEGF receptor-3 (VEGFR-3, Flt-4) [44, 45]. It remains to be determined whether VEGF-C and VEGF-D along with VEGFR-3 play a role in lymphangiogenesis during bladder cancer development. Another interesting line of research involves LYVE-1-positive tumor associated macrophages  that have indeed been associated with tumor lymphangiogenesis [47, 48]. In the eye, a stepwise mechanism of inflammation-associated de novo lymphangiogenesis involves potential lymphatic progenitor cells  derived from circulation that transmigrate through the connective tissue stroma, presumably in the form of macrophages [49–51], and finally incorporate into the growing lymphatic vessels . Our present findings indicate that in addition to LYVE-1-positive lymphatic endothelial cells, during bladder cancer development, LYVE-1 positive macrophages were found in the bladder detrusor muscle isolated from SV40- lacZ mice. Although this may be only a circumstantial finding, it opens a testable hypothesis on the role of macrophages and other inflammatory cells in bladder lymphangiogenesis. The introduction of SV40- lacZ along with the visualization and quantification techniques described here will permit further investigation on this subject.
It has been proposed that lymphangiogenesis is correlated with tumor metastasis. Increasing knowledge of the tumor's biological significance in lymphatics within the tumors (intratumoral lymphatics, ITLs) and at the tumor periphery (peritumoral lymphatics, PTLs) has greatly promoted understanding of tumor access into the lymphatic system by inducing lymphangiogenesis or by co-opting preexisting lymphatics . Indeed, peritumoral lymphatics have also been associated with both regional metastasis and survival in bladder , lung , breast , and prostate cancer . But the question still remains as to whether pre-existing vessels are sufficient to serve this function, or whether tumor cell dissemination requires de novo lymphatic formation or an increase in lymphatic size. In this regard, Fernandez and collaborators reported that in the human bladder, higher intratumoral LVD correlates significantly with poor histological differentiation, and that higher peritumoral LVD showed a significant association with the presence of lymph node metastasis . Although peritumoral lymphatic vessels contribute to tumor metastasis, opposite views exist as to whether intratumoral lymphatics have any role in tumor metastasis [56, 57]. Padera and collaborators examined functional lymphatics associated with mouse tumors expressing normal or elevated levels of VEGF-C . Although VEGF-C over-expression increased lymphatic surface area in the tumor margin and lymphatic metastasis, these tumors contained no functional lymphatics, as assessed by four independent functional assays and IHC staining . These findings suggest that the functional lymphatics in the tumor margin alone are sufficient for lymphatic metastasis and should be targeted therapeutically . Our MRI results are in agreement with those described by Pandera and collaborators  in the sense that intratumor lymphatic vessels have a reduced function when compared to normal areas of the bladder.
Another question answered by the present work was whether an increase in the number of lymphatic vessels leads to increased function. For this purpose, we followed the strategy recently reviewed by Neeman and collaborators . The contrast agent introduced here was originally described by Dafni and collaborators [14–16], and subsequently by Pathak and collaborators , for visualization of lymphatics and determination of their function. The modification of conjugating biotin-BSA-Gd-DTPA  to Cy5.5 permitted us to use NIRF and MRI to follow the dynamics of the same compound and calculate lymphatic vessel function. The advantage of using NIRF is the faster time for data acquisition. NIRF information permitted us to narrow the number of time points for subsequent MRI studies.
The proposed mechanism of visualization of BSA-Gd-Cy5.5 (~82 kDa) is based on the presence of BSA which prolongs its lifetime in circulation. Initially the probe is confined to blood vessels and it is systemically distributed, and as the time passes it extravasates to the extracellular space in points of increased vascular permeability. Both NIRF and MRI are able to detect when BSA-Gd-cy5.5 starts to accumulate in the extracellular space and when its accumulation reaches a plateau. This was done at the MRI level by following the longitudinal relaxation rates (1/T1) that correlates with increased uptake of the Gd-probe from the blood vessels into the urinary bladder extra vascular space. This phase was called the "early phase" in this manuscript. After 80 minutes of the plateau, a second series of MRI were started and followed the clearance of BSA- Gd-Cy5.5 from the extravascular by the lymphatic vessels. This was supported by ex vivo images which indicated that, at this point, most of the BSA-Gd-Cy5.5 is drained from the tissue by lymphatic vessels.
Although MRI lymphangiography does measure areas of draining and pooling instead of directly evaluating lymphatic vessel function, the delayed enhancement observed at later time points from the Gd-probe may also reflect uptake of Gd-probe in the lymphatics. In addition, this information can not be obtained any other way and clinical studies attest the validity of using lymphangiography for assessment of lymphatic vessel function [60–64]. A step forward in this direction was introduced here by the use of a single probe that can be imaged by MRI and NIRF. The first indicates areas of draining and pooling and the second permitted the temporal association of images with cross-sections indicating the relative distribution of the probe between CD31-positive blood vessels and LYVE-1-positive lymphatic vessels.
As pointed out by Neeman and collaborators , contrast MRI data are typically dominated by vascular permeability, which often masks the relatively slow lymphatic drain. To separate vascular leakage from the lymphatic drain, an avidin chase needs to be introduced [14, 16] which through the rapid clearance of intravenously administered biotin-BSA-Gd-DTPA, allowed them to experimentally track interstitial convection and lymphatic drain in the absence of continuing vascular leakage [14, 16]. Our present MRI results are not corrected for vascular leakage. Therefore, future experiments will take into consideration this point for a more detailed analysis of lymphatic vessel function using the advantage of the presence of biotin in the Gd-Cy5.5 molecule.
Another advantage of NIRF was to permit the collection of tissues exhibiting fluorescence. In this regard, we were able to further pursue the morphology of lymph sacs using dextran-Cy5.5. This will allow us in the near future to determine the contractility of lymph sacs and abdominal lymphatic vessels. The rhythmic activity observed in the abdominal lymphatics draining the urinary bladder is in agreement with recent results that indicated that mesenteric lymphatic vessels have a true pacemaker mechanism [65, 66]. The experiment described in Figures 10, 11, 12 will permit the capture and evaluation of large lymphatic function in health and disease.