Microvascular fluid exchange (flow J(v)) underlies plasma/interstitial fluid (ISF) balance and oedematous swelling. The traditional form of Starling's principle has to be modified in light of insights into the role of ISF pressures and the recognition of the glycocalyx as the semipermeable layer of endothelium. Sum-of-forces evidence and direct observations show that microvascular absorption is transient in most tissues; slight filtration prevails in the steady state, even in venules. This is due in part to the inverse relation between filtration rate and ISF plasma protein concentration; ISF colloid osmotic pressure (COP) rises as J(v) falls. In some specialized regions (e.g. kidney, intestinal mucosa), fluid absorption is sustained by local epithelial secretions, which flush interstitial plasma proteins into the lymphatic system. The low rate of filtration and lymph formation in most tissues can be explained by standing plasma protein gradients within the intercellular cleft of continuous capillaries (glycocalyx model) and around fenestrations. Narrow breaks in the junctional strands of the cleft create high local outward fluid velocities, which cause a disequilibrium between the subglycocalyx space COP and ISF COP. Recent experiments confirm that the effect of ISF COP on J(v) is much less than predicted by the conventional Starling principle, in agreement with modern models. Using a two-pore system model, we also explore how relatively small increases in large pore numbers dramatically increase J(v) during acute inflammation.

This review examined the relation between interstitial hydraulic permeability and chemical composition, using porous matrix theory, and assessed the view that interstitial conductivity is governed by GAG concentration. Conductivity correlates negatively with both GAG and collagen concentration over a wide variety of tissues. Conductivities of GAG matrices in vitro, coupled with other quantitative considerations, indicate, however, that no single class of fixed fibrous element exists at a sufficiently high concentration to account by itself for the low conductivity in most tissues. It seems that the low interstitial conductivity arises from the combined interactive effects of three main classes of fibrous element: collagen fibrils, GAG and proteoglycan core protein. In most cases the proteoglycan complex plays a major role which is significantly amplified, however, by the collagen fibril network.

The mechanisms that cause tumors such as melanomas to metastasize into peripheral lymphatic capillaries are poorly defined. Non-mutually-exclusive mechanisms are lymphatic endothelial cell (LEC) chemotaxis and proliferation in response to tumor cells (chemotaxis-lymphangiogenesis hypothesis) or LECs may secrete chemotactic agents that attract cancer cells (chemotactic metastasis hypothesis). Using migration assays, we found evidence supporting both hypotheses. Conditioned medium (CM) from metastatic malignant melanoma (MMM) cell lines attracted LEC migration, consistent with the lymphangiogenesis hypothesis. Conversely, CM from mixed endothelial cells or LECs, but not blood endothelial cells, attracted MMM cells but not non-metastatic melanoma cells, consistent with the chemotactic metastasis hypothesis. MMM cell lines expressed CCR7 receptors for the lymphatic chemokine CCL21 and CCL21 neutralizing antibodies prevented MMM chemotaxis in vitro. To test for chemotactic metastasis in vivo tumor cells were xenotransplanted into nude mice B1 cm from an injected LEC depot. Two different MMM grew directionally towards the LECs, whereas non-metastatic melanomas did not. These observations support the hypothesis that MMM cells grow towards regions of high LEC density owing to chemotactic LEC secretions, including CCL21. This chemotactic metastasis may contribute to the close association between metastasizing tumor cells and peritumor lymphatic density and promote lymphatic invasion.

Synovial fluid lubricates joints and tendon sheaths, and carries metabolites to and from the avascular articular cartilage. The fluid is generated primarily by ultrafiltration of synovial microvascular plasma into the joint cavity when intra-articular pressure is low, as in extension. Hyaluronan is added to the fluid by actively secreting synoviocytes in the lining. The role of hyaluronan as a hydrodynamic lubricant has long been recognized. A second hydraulic role was suggested recently, namely the conservation of intraarticular fluid during joint flexion. This idea arose from the observation that, as intra-articular pressure is raised, the opposition to fluid drainage from the joint cavity increases if hyaluronan is present, but not when it is absent (McDonald & Levick, 1995). The present study explores new facets of this action and its mechanism. Hyaluronan is a non-sulphated glycosaminoglycan chain of variable length. Native chains in synovial fluid have a molecular mass of (2-7) ² 10É Da. In some commercial preparations, however, the molecular mass can be an order of magnitude smaller, and this was the case with the umbilical cord hyaluronan studied previously (McDonald & Levick, 1995). Despite this, umbilical hyaluronan greatly reduces the trans-synovial escape of fluid from the joint cavity in rabbit knees, and alters the fundamental shape of the pressure-flow relation. In the absence of hyaluronan, the relation between intra-articular pressure and trans-

Malignant melanoma (MM), the most common cause of skin cancer deaths, metastasises to regional lymph nodes. In animal models of other cancers, lymphatic growth is associated with metastasis. To assess if lymphatic density (LD) was increased in human MM, and its association with metastasis, we measured LD inside and around archival MM samples (MM, n ¼ 21), and compared them with normal dermis (n ¼ 11), basal cell carcinoma (BCC, n ¼ 6) and Merkel cell carcinoma (MCC), a skin tumour thought to metastasise through a vascular route (MCC, n ¼ 6). Lymphatic capillary density (mm À2 ), as determined by immunohistochemical staining with the lymphatic specific marker LYVE-1, was significantly increased around MM (10.072.5 mm À2 ) compared with normal dermis (2.470.9 mm À2 ), BCC (3.070.9 mm À2 ) and MCC (2.471.4 mm À2 ) (Po0.0001). There was a small decrease in LD inside MM (1.170.7 mm À2 ) compared with normal dermis, but a highly significant decrease in BCC (0.1470.13) and MCC (0.1272.4) (Po0.01 Kruskal -Wallis). Astonishingly, LD discriminated between melanomas that subsequently metastasised (12.871.6 mm À2 ) and those that did not (5.471.1 mm À2 , Po0.01, Mann -Whitney). Lymphatic invasion by tumour cells was seen mainly in MM that metastasised (70% compared with 12% not metastasising, Po0.05 Fisher's Exact test). The results show that LD was increased around MMs, and that LD and tumour cell invasion of lymphatics may help to predict metastasis. To this end, a prognostic index was calculated using LD, lymphatic invasion and thickness that clearly discriminated metastatic from nonmetastatic tumours.

Axillary surgery for breast cancer may be followed, months to years later, by chronic arm lymphedema. A simple 'stopcock' mechanism (reduced lymph drainage from the entire limb through surviving lymphatics) does not explain many clinical aspects, including the delayed onset and selective sparing of some regions, e.g., hand. Quantitative lymphoscintigraphy reveals that lymph drainage is slowed in the subcutis, where most of the edema lies, and in the subfascial muscle compartment, which normally has much higher lymph flows than the subcutis. Although the muscle does not swell significantly, the impaired muscle drainage correlates with the severity of arm swelling, indicating a likely key role for muscle lymphatic function. A new method, lymphatic congestion lymphoscintigraphy, showed that the edema is associated with a reduced contractility of the arm lymphatics; the weaker the active lymphatic pump, the greater the swelling. Delayed lymphatic pump failure may result from chronic raised afterload, as in hypertensive cardiac failure, and may account for the delayed onset of swelling. A further novel finding is that lymph flow is raised in both the subcutis and muscle of both arms in postsurgical breast patients who later developed breast cancer-related lymphedema (BCRL), compared with patients who did not develop BCRL. This new observation indicates a predisposition to BCRL in some women. Further evidence for predisposing abnormalities is the finding of lymphatic abnormalities in the contralateral (nonswollen) arm in women with established BCRL. Such predisposing factors could explain why some women develop BCRL after sentinel node biopsy, whereas others do not after clearance surgery. Future research must focus on prospective observations made from before surgery until BCRL develops.

1. The flow of Ringer solution or paraffin oil from an infusion reservoir into the cavity of the knee (stifle) joint was measured in anesthetized rabbits, as intraarticular pressure was progressively elevated from its intrinsic slightly subatmospheric value to +25 cm H2O. 2. Paraffin oil did not penetrate the tissues lining the joint cavity, yet a continuous flow of oil occurred into the joint at pressures over +2 cm H2O. It was concluded that the joint investment behaved as a visco‐elastic tissue. 3. Trans‐synovial flow of Ringer solution was calculated by correcting the observed inflow for visco‐elastic expansion of the joint capsule. At intra‐articular pressures +2 to +9 cm H2O, trans‐synovial flow increased at an average rate of 0.49 microliter min‐1.cm H2O‐1. The hydraulic conductivity of the synovium was therefore similar to that of subcutaneous connective tissue. At around +9 cm H2O, the 'breaking pressure', the slope of the pressure‐flow relationship increased by almost sixfold to 2.81 microliter min‐1.cm H2O‐1. 4. Changes in joint visco‐elasticity, synovial surface area, blood pressure, colloid osmotic pressure of plasma and of joint fluid, and inflammation were excluded as explanations of the marked increase in rate of fluid absorption, which is tentatively attributed to increases in synovial hydraulic conductivity. Some physiological and clinical implications of the data are discussed.

Background— Mutations in the FOXC2 gene cause lymphedema distichiasis, an inherited primary lymphedema in which a significant number of patients have varicose veins. Because lymphedema distichiasis is believed to be caused by lymphatic valve failure (reflux), and FOXC2 is highly expressed on venous valves in mouse embryos, we tested the hypothesis that FOXC2 mutations may be linked to venous valve failure and reflux. Methods and Results— The venous system of the leg was investigated with Duplex ultrasound. Pathological reflux was recorded by color Duplex ultrasound in all 18 participants with a FOXC2 mutation, including 3 without lymphedema. Every participant with a mutation in FOXC2 showed reflux in the great saphenous vein (n=18), compared with only 1 of 12 referents (including 10 family members; P <0.0001, Fisher exact test). Deep vein reflux was recorded in 14 of 18 participants. Conclusions— FOXC2 is the first gene in which mutations have been strongly associated with primary venous valve failure in both the superficial and deep veins in the lower limb. This gene appears to be important for the normal development and maintenance of venous and lymphatic valves.