A robust control system for targeting melanoma by a supermolecular DDMC/paclitaxel complex.

A DEAE-dextran-MMA copolymer (DDMC)-paclitaxel (PTX) conjugate was prepared using PTX as the guest and DDMC as the host. The resistance of B16F10 melanoma cells to PTX was confirmed, while the DDMC-PTX conjugate showed excellent anticancer activity that followed the Hill equation. The robustness in the tumor microenvironment of the allosteric system was confirmed via BIBO stability. This feedback control system, explained via a transfer function, was very stable and showed the sustainability of the system via a loop, and it showed superior anti-cancer activity without drug resistance from cancer cells. The block diagram of this signal system in the tumor microenvironment used its loop transfer function G(s) and the dN(s) of the external force. This indicial response is an ideal one without a time lag for the outlet response. The cell death rate of DDMC-PTX is more dependent on the Hill coefficient n than on the Michaelis constant Km. This means that this supermolecular reaction with tubulin follows an "induced fit model".

Lymph node lymphangiogenesis: a new concept for modulating tumor metastasis and inflammatory process.

The proliferation of lymphatic endothelial cells (LECs) occurs not only in tumor and inflamed tissues, but also in regional draining lymph nodes (LNs). The lymph node lymphangiogenesis (LNLG) has recently emerged as a prominent area in biomedical research, because it is involved in the pathogenesis of several human diseases. The LEC functional features and lymphatic remodeling regulated by lymphangiogenic factors actively promote tumor metastasis and the inflammation process. VEGF-A/VEGFR-2 and VEGF-C/-D/VEGFR-3 have been implicated as the prime mediators in inflammation- or tumor-induced LNLG. This knowledge may provide a foundation for further understanding of specific modification in the gene expression, cell migration, and differentiation of LECs and other cells in lymphatic-associated diseases. Importantly, it should be taken into consideration that inflammation and lymphangiogenesis are strongly linked in the formation and metastasis of cancer when designing therapeutic strategies.

Lymphatic endothelial cells, inflammatory lymphangiogenesis, and prospective players.

De novo lymphangiogenesis influences different pathological courses via modulating tissue fluid homeostasis, macromolecule absorption, and leukocyte transmigration. During the past decade, improved understanding of lymphatic biology, especially VEGF-C/-D/VEGFR-3-mediating lymphangiogenesis has substantially promoted clinical research in lymphatic insufficiency. The role of lymphangiogenesis in the setting of the inflammatory processes observed in transplants, inflamed cornea, wound healing, acquired lymphedema and tumor invasion, however, remains to be elucidated. The chemokine family of peptide chemoattractants and other mediators, e.g., CCL21-CCR7, D6, NF-kappaB and TNF-alpha may be important contributors to pathophysiological changes of lymphatic endothelial cells (LECs) and inflammatory lymphangiogenesis. Dendritic cells and macrophages may also involve in LEC proliferation and differentiation. Increased knowledge of LEC-specific modulators in inflammatory microenvironment is vital for prevention and treatment of lymphatic-associated diseases.

Investigation of intratumoural and peritumoural lymphatics expressed by podoplanin and LYVE-1 in the hybridoma-induced tumours.

Tumour-associated lymphatics contribute to a key component of metastatic spread, however, the biological interaction of tumour cells with intratumoural and peritumoural lymphatics (ITLs and PTLs) has remained unclear. To address this important issue, we have focused on the morphological and molecular aspects of newly formed lymphatics (lymphangiogenesis) and pre-existing lymphatics in the intratumoural and peritumoural tissues by using a hybridoma-induced tumour model. In the present study, ITLs with very high vessel density within the tumour mass showed small and flattened contours that varied from non-solid-to-solid tumours, whereas PTLs were relatively disorganized and tortuous, and packed with a cluster of tumour cells at the tumour periphery. Lymphatic endothelial cells (LECs) both in ITLs and PTLs were expressed with LYVE-1 and podoplanin in various tumour tissues, in which initial lymphatics were extremely extended and dilated. The tumour cells were frequently detected adhering to or penetrating lymphatic walls, especially near the open junctions. In the metastatic tissues, lymphangiogenic vasculatures occurred within the tumour matrix, and collecting PTLs represented abnormal twisty valve leaflets. The Western blot and RT-PCR analysis showed local variations of LEC proliferating potentials and lymphatic involvement in metastasis by a distinct profile of the protein and mRNA expression by LYVE-1, podoplanin, Prox-1 and vascular endothelial growth factor-3 (VEGFR-3). These findings indicated that both ITLs and PTLs, including enlarged pre-existing and newly formed lymphatics, may play a crucial role in metastasis with an active tumour cell adhesion, invasion, migration and implantation.

Characteristics of lymphatic endothelial cells in physiological and pathological conditions.

Impairment of lymphatic structure and function, e.g., inadequate endothelial permeability and intercellular openings, abnormal lymphangiogenesis and overexpression for immunoreactive agents, will result in tumor metastasis, autoimmune response alteration and accumulation of interstitial fluid and proteins. Recently, several novel molecules have been identified that allow a more precise distinction between lymphatic and blood vascular endothelium. The differences in expression of endothelial markers on the lymphatic vessel strongly suggest the possibility that there will be important divergence in the differentiating and regenerating responses in lymphatic behavior to various pathological processes. Undoubtfully, molecular techniques would also lead to the definition of unique markers found on lymphatic endothelial cells (LECs) in lymphatic-associated diseases which are mostly involved in lymphangiogenesis. This review is mainly concentrated on the characteristics of LECs in diabetes, wound healing, lymphedema and tumor, especially in the experimental models that have offered insight into the LEC role in these diseases affecting the lymphatic system. Increased knowledge of the molecular signaling pathways driving lymphatic development and lymphangiogenesis should boost the impact of therapeutics on the diseases. Although the field about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels has experienced rapid progress in the past few years, an understanding of the basis of the differences and their implications in the pathological conditions will require much more investigation.

Histochemical analysis of lymphatic endothelial cells in the pancreas of non-obese diabetic mice.

We studied the relationship between insulitic development and function-structural changes of pancreatic lymphatics in non-obese diabetic (NOD) mice using combined 5'-nucleotidase (5'-Nase) enzyme histochemical and secondary lymphoid tissue chemokine (SLC/CCL21) immunohistochemical methods. Interlobular lymphatic vessels were positive for 5'-Nase throughout the pancreas, and dependent on both blood vessels and pancreatic ducts. Intralobular initial lymphatics were rare and occasionally ran in the neighbourhood of islets. During the non-insulitic stage, the 5'-Nase-reactive product was evenly distributed on the surface of lymphatic endothelial cells (LECs) with weak expression of CCL21. The activity of 5'-Nase on lymphatic vessels became slightly reduced as insulitis developed. The increasing blood glucose values appeared to be consistent with an increasing CCL21 expression by the endothelial lining, especially on the surface of LECs adjacent to the infiltrated islets and tissues. Lymphocytes and dendritic cells (DCs) were frequently located in the connective tissue, surrounding the lymphatic wall with deposition of 5'-Nase precipitates. As the infiltration became severe, lymphocytes and DCs accumulated within lymphatic vessels and expressed high levels of CCL21. The most significant finding was that many DCs adhered to lymphatic vessels, transmigrating via the thin and indented endothelial walls. The activity of 5'-Nase was increased on the adhesion surface between DCs (or lymphocytes) and LECs. The latter were characterized by open intercellular junctions and obvious cytoplasmic protrusions. These results suggest that LECs closely interact with DCs and lymphocytes, and play a key role in the migration of DCs and lymphocytes via lymphatic vessels during the pathological processes of insulitis in NOD mice.

Histochemical analysis of lymphatic endothelial cells in lymphostasis.

The ultrastructure of endothelial cells of intestinal lymphatics and the thoracic duct (TD) and the relation to lymphostasis were examined in rats and monkeys. Localization of 5'-nucleotidase (5'-Nase) and endothelial nitric oxide synthase (eNOS) was studied. In normal lymphatic endothelial cells, 5'-Nase reaction product was evenly deposited on the cell surface in vivo and on cultured TD endothelial cells (TDECs), whereas eNOS was evenly distributed throughout the nucleus and cytoplasm. TDECs had a long filamentous process extending towards the subendothelial extracellular matrix but became flat and regular within 30-40 minutes after gastric perfusion with olive oil. According to their electron-density, two types of cells were found in the TD endothelial layer. The cells with low electron-density exhibited stronger 5'-Nase activity. Valves were bicuspid formations and the valvular endothelial surface of the convex side showed weaker 5'-Nase activity than the concave side. During TD blockage-induced lymphostasis in rats, the 5'-Nase product was almost not discernible in the TDECs within 2 weeks. Larger vesicles were found in the endothelial cytoplasm of the ligated TD. Their number decreased after 6-12 weeks. The small intestinal lymphatics in the mucosa and submucosa were dilated, with numerous open intercellular junctions. The endothelial lining appeared to have reduced activities for 5'-Nase and eNOS in 9 of 11 experimental animals. The results indicated that the inability of the open intercellular junctions, normally working as one-way endothelial flap valves, may be a key morphological feature after TD blockage. Reduced eNOS and 5'-Nase may functionally influence contractile activity and transport capability of the lymphatic vessels in the lymphostasis.

Structural organization and fine distribution of lymphatic vessels in periodontal tissues of the rat and monkey: a histochemical study.

The structural organization and fine distribution of lymphatic vessels in the periodontal tissues (gingiva, periodontium and alveolar process) were examined by light and electron microscopy using an enzyme-histochemical method. Whole mount preparations of periodontal membranes peeled from the teeth and cryostat sections of normal or decalcified tissues treated with EDTA were double-stained using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) and examined by light microscopy. This staining procedure allowed the lymphatic vessels in the periodontal tissue to be differentiated from blood vessels. Well-developed 5'-Nase-positive lymphatics were observed in the gingiva and periodontium. The histochemical aspects of 5'-Nase activity in lymphatic vessels are discussed in detail, with special reference to the supply of Mg++ ions. A network of 5'-Nase-positive lymphatics was observed in whole mount preparations of the periodontal membrane for the first time. This network was also observed in the tissue sections. More 5'-Nase-positive lymphatics were seen in the root area of the periodontium than in the cervical area. 5'-Nase-positive lymphatics in residual tissue blocks remaining after cryostat sectioning and in whole mount preparations were highlighted with good contrast and resolution on backscattered electron images produced by scanning electron microscopy. Dense granular precipitations resulting from the 5'-Nase reaction were observed on the luminal surface of the lymphatic endothelial cells as well as on the basal side but were absent in the blood vessels.

Intrinsic interrelation of lymphatic endothelia with nerve elements in the monkey urinary bladder.

Histochemical staining techniques for 5'-nucleotidase (5'-Nase) and acetylcholinesterase (AChE) were undertaken to localize the lymphatic network and nerve plexus in the monkey urinary bladder. Abundant 5'-Nase-positive lymphatic networks were characterized by increased number of valve-like structures and decreased calibre of blind-ends from the subepithelium to the subserosa. AChE-positive nerve fibers were visible throughout the vesical walls as fine plexuses, the densest being the neuromuscular plexus among the detrusor muscle cells or in each muscle bundle. AChE-positive nerve fibers or terminals were more frequently discernible around blood vessels than around lymphatics, and showed more intimate association with the lymphatics in the muscularis than those in the subepithelium. The nerve terminals in the subepithelium were frequently separated from attenuated lymphatic endothelium by the long processes of fibroblasts or some connective tissue cells. An ultrastructural observation revealed that unmyelinated nerve fibers with numerous neurofilaments and neurotubules run in close apposition to the lymphatic endothelium. Noteworthily, fewer terminal varicosities containing numerous small agranular vesicles (30-50 nm) and mitochondria, partially or completely bare of their Schwann cell covering in the vicinity of the lymphatic endothelium, were found in the subendothelium of initial lymphatics than in collecting ones. These terminals were occasionally identified at a distance of 120-350 nm from the subendothelial aspect of valve-originating roots, although no direct innervation of the vascular muscle cells could be found. A loose fibro-elastic connective tissue was usually interlaced between glial cell covering and lymphatic endothelium. The intrinsic interrelation of the lymphatic wall with the nerve plexus implies that the twisted subendothelial nerve terminals might be involved in intramural lymph drainage of the bladder.

Enzyme-histochemical study on postnatal development of rat stomach lymphatic vessels.

Postnatal development of rat gastric lymphatics was studied by an enzyme-histochemical method to elucidate the morphological changes of lymphatics and their relationship to maturation and function, especially in the glandular portion. The significant features of 5'-Nase-positive lymphatics in distribution and structure were examined in different stages (within 24 hr, 4-21 days, and 2 months). Lymphatics in the greater curvature and anterior wall grew much slower than those in the lesser curvature and posterior wall of the stomach in newborn and infant rats. Lymphatic islands isolated from the primary lymphatic networks in the submucosa and subserosa underwent a morphological change during this early period. This is considered one of the basic steps in lymphatic development. Occurrence of lymphatic networks in the deep lamina propria indicates that development in the gastric wall is well characterized from Day 10. With further growth and modification of lymphatics, the networks in the different layers formed an extensive communication network and many lymphatic valves were found in the submucosa and subserosa. Pinocytotic vesicles, open junctions, and intraendothelial channels were frequently detected in the mucosal and submucosal lymphatic networks of the corpus-antrum and antrum-duodenum divisional zones in the adult rats. These findings suggest that developing lymphatics in the rat stomach may represent rapidly growing tissue not only with high 5'-Nase activity but also with high adaptability for future physiological demands.

Demonstration of the intralobular lymphatics in the guinea pig pancreas by an enzyme-histochemical method.

Intralobular lymphatics in the guinea pig pancreas were demonstrated enzyme-histochemically showing the extent, distribution and fine structure by combined light and transmission electron microscopy. 5'-nucleotidase(5'-Nase)-positive lymphatic vessels were present throughout the pancreas. Intralobular lymphatics among the acini were comparatively rare and generally independent of the blood capillaries, pancreatic ducts and acini. These lymphatics revealed the usual structural features, such as typical intercellular junctions and very tenuous vascular walls without continuous basal laminae. Fine precipitates of the cerium-based reaction product for 5'-Nase activity were found to be associated with cell membranes of the lymphatic endothelium and pinocytotic vesicles. Lymphatics were not closely related to the endocrine islets, although alkaline phosphatase(ALPase)-positive blood capillaries were well developed. Collecting lymphatic vessels with valves with weaker 5'-Nase activity were also detected in the interlobular connective tissue. ALPase activity, absent in the lymphatics, was positive in the blood capillaries, suggesting that it is also a useful way demonstrating, histochemically, the blood capillaries in the guinea pig pancreas.

Afferent migration of the Kurloff cells via lymphatics into the thymus of estradiol-treated guinea pigs.

The spatial distribution and migration of Kurloff cells containing PAS-positive large inclusion bodies in the thymus of estradiol-treated guinea pigs were histochemically studied by a combination of light and electron microscopy. Male guinea pigs were examined at various intervals from 7 days to 3 months after a single subcutaneous injection of estradiol. Differentiation of lymphatics from blood capillaries was performed by a 5'-nucleotidase (5'-Nase) staining method and the occurrence of Kurloff cells within 5'-Nase-positive lymphatics was confirmed by ultrastructural histochemistry. Several Kurloff cells first appeared at 7 days within lymphatics in the thymic capsule or interlobular connective tissues. At 12-15 days after estradiol administration, a lymphatic accumulation, a so-called "lymphatic center", was seen in the thymic septa even though few Kurloff cells were present within the thymic parenchyma. The "lymphatic center" contained many Kurloff cells located in its periphery and in the surrounding marginal sinus which communicated with the thymic interlobular lymphatics. At 21 days after estradiol, Kurloff cells were preferentially accumulated along the corticomedullary junction extravascularly. Later the distribution was more diffuse. The conspicuous accumulation of Kurloff cells in the corticomedullary region could reflect an inability of Kurloff cells to use blood vessels as a route for migration. These findings strongly suggest the afferent migration of Kurloff cells into the thymus via lymphatics.

The distribution and architecture of lymphatic vessels in the rat stomach as revealed by an enzyme-histochemical method.

Enzyme-histochemical methods of staining for 5'-nucleotidase (5'-Nase) and alkaline phosphatase (ALPase) were successfully applied to study the distribution and architecture of lymphatic vessels and their relationships to blood vessels in the rat stomach. Extensively lymphatic capillary networks were found in the gastric wall, but there were significant differences in their extent, pattern, distribution and structure in the four different zones: esophagus-stomach (E-S), forestomach-corpus (F-C), corpus-antrum (C-A) and antrum-duodenum (A-D). 5'-Nase-ALPase double staining revealed that the 5'-Nase-positive lymphatic vessels run in close proximity to ALPase-positive arteries and veins. The fine blood capillary network was located superficially to the lymphatic network within the same layer in the gastric wall. The abundant lymphatic network located in the deep lamina propria and the lamina muscularis mucosa was always closely associated with the base of the lowest gastric glands, and yet no interglandular lymphatic capillaries were encountered in the corpus or antrum. In contrast, fewer lymphatic capillaries were present in the lamina propria beneath the squamous epithelium of the forestomach. The distribution of the well-developed lymphatic networks with valve-like structures in the submucosa and subserosa exhibited typical features, i.e., the distribution was annular in the submucosa and fan-shaped in the subserosa in the antrum near the duodenum. Open junctions of lymphatic endothelial cells were seen in the deep lamina propria and submucosa. Collecting lymphatics containing valves were mainly located deep in the submucosa and subserosa. The deep lamina propria and submucosa may play a key role in lymph formation and interstitial tissue fluid homeostasis as well as in pathological processes in certain diseases. The present findings obtained by interstitially injecting ultra-fine carbon particle suspensions or Evans blue showed that a great deal of lymph drained into the lymphatics accompanying the left gastric artery. The existence of a forestomach may explain the complicated organization and constitution of lymphatic networks in the rat stomach.

Enzyme triple staining for differentiation of lymphatics from venous and arterial capillaries.

5'-nucleotidase (5'-Nase)-dipeptidyl aminopeptidase IV (DAPase)-alkaline phosphatase (ALPase) triple staining was used to differentiate lymphatics from venous and arterial capillaries in a variety of mammalian tissue sections including human. This triple staining method facilitates specific identification under a light microscope of 5'-Nase activity in lymphatics, DAPase activity in venous capillaries and venules and ALPase activity in arterial capillaries and arterioles. This technique depicts initial lymphatics more clearly and extensively than other methods so far reported although some interspecies and tissue differences are obtained in each enzyme activity.