Ionic currents in intimal cultured synoviocytes from the rabbit.

Hyaluronan, a joint lubricant and regulator of synovial fluid content, is secreted by fibroblast-like synoviocytes lining the joint cavity, and secretion is greatly stimulated by Ca(2+)-dependent protein kinase C. This study aimed to define synoviocyte membrane currents and channels that may influence synoviocyte Ca(2+) dynamics. Resting membrane potential ranged from -30 mV to -66 mV (mean -45 ± 8.60 mV, n = 40). Input resistance ranged from 0.54 GΩ to 2.6 GΩ (mean 1.28 ± 0.57 GΩ; ν = 33). Cell capacitance averaged 97.97 ± 5.93 pF. Voltage clamp using C(s+) pipette solution yielded a transient inward current that disappeared in Ca(2+)-free solutions and was blocked by 1 µM nifedipine, indicating an L-type calcium current. The current was increased fourfold by the calcium channel activator FPL 64176 (300 nM). Using K(+) pipette solution, depolarizing steps positive to -40 mV evoked an outward current that showed kinetics and voltage dependence of activation and inactivation typical of the delayed rectifier potassium current. This was blocked by the nonspecific delayed rectifier blocker 4-aminopyridine. The synoviocytes expressed mRNA for four Kv1 subtypes (Kv1.1, Kv1.4, Kv1.5, and Kv1.6). Correolide (1 µM), margatoxin (100 nM), and α-dendrotoxin block these Kv1 subtypes, and all of these drugs significantly reduced synoviocyte outward current. The current was blocked most effectively by 50 nM κ-dendrotoxin, which is specific for channels containing a Kv1.1 subunit, indicating that Kv1.1 is critical, either as a homomultimeric channel or as a component of a heteromultimeric Kv1 channel. When 50 nM κ-dendrotoxin was added to current-clamped synoviocytes, the cells depolarized by >20 mV and this was accompanied by an increase in intracellular calcium concentration. Similarly, depolarization of the cells with high external potassium solution caused an increase in intracellular calcium, and this effect was greatly reduced by 1 µM nifedipine. In conclusion, fibroblast-like synoviocytes cultured from the inner synovium of the rabbit exhibit voltage-dependent inward and outward currents, including Ca(2+) currents. They thus express ion channels regulating membrane Ca(2+) permeability and electrochemical gradient. Since Ca(2+)-dependent kinases are major regulators of synovial hyaluronan secretion, the synoviocyte ion channels are likely to be important in the regulation of hyaluronan secretion.

Signal pathways regulating hyaluronan secretion into static and cycled synovial joints of rabbits.

Joint lubrication, synovial fluid conservation and many pathophysiological processes depend on hyaluronan (HA). Intra-articular HA injection and exercise, which stimulates articular HA production, ameliorate osteoarthritis. We therefore investigated the pathways regulating movement-stimulated articular HA secretion rate ( ) in vivo. Endogenous HA was removed from the knee joint cavity of anaesthetised rabbits by washout. Joints were then cycled passively or remained static for 5 h, with/without intra-articular agonist/inhibitor, after which newly secreted HA was harvested for analysis. Movement almost doubled . Similar or larger increases were elicited in static joints by the intra-articular Ca(2+) ionophore ionomycin, prostaglandin E(2), cAMP-raising agents, serine/threonine phosphatase inhibitor and activation of protein kinase C (PKC). PKC-stimulated secretion was inhibited by the PKC inhibitor bisindolylmaleimide I and inhibitors of the downstream kinases MEK-ERK (U0126, PD98059). These agents inhibited movement-stimulated secretion of HA (MSHA) only when the parallel p38 kinase path was simultaneously inhibited by SB203580 (ineffective alone). The phospholipase C inhibitor U73122 almost fully blocked MSHA (P = 0.001, n = 10), without affecting static . The ENaC channel blocker amiloride inhibited MSHA, whereas other inhibitors of stretch-activated channels (Gd(3+), ruthenium red, SKF96365) did not. It is proposed that MSHA may be mediated by PLC activation, leading to activation of parallel PKC-MEK-ERK and p38 kinase pathways.

Mechanosensitive hyaluronan secretion: stimulus-response curves and role of transcription-translation-translocation in rabbit joints.

Joint movement was recently shown to stimulate the secretion of the lubricant hyaluronan (HA); also, exercise therapy and intra-articular hyaluronan injections are used to treat moderate osteoarthritis. The present study quantifies the stimulus-response curves for HA secretion in vivo and reports a role of transcription-translation-translocation in the secretory response. After washing out endogenous HA from anaesthetized, cannulated rabbit knees, the joints were cycled passively at various frequencies and durations, with or without intra-articular inhibitors of protein synthesis and Golgi processing. Newly secreted HA was harvested for analysis after 5 h. Joints displayed graded, non-linear stimulus-response curves to both duration and frequency of movement; 1 min duration per 15 min or a frequency of 0.17 Hz raised HA secretion by 42-54%, while rapid (1.5 Hz) or prolonged cycling (9 min per 15 min) raised it by 110-130%. Movement-stimulated secretion and phorbol ester-stimulated secretion were partly inhibited by the translation inhibitor cycloheximide, by the transcription-translation inhibitors actinomycin D and puromycin and by the Golgi translocation inhibitor brefeldin A. There is thus a graded coupling between HA secretion and cyclic joint movement that depends partly on new protein synthesis. This is likely to be important for joint homeostasis, providing protection during repetitive cycling and potentially contributing to exercise therapy for osteoarthritis.

Chemokine-mediated migration of melanoma cells towards lymphatics--a mechanism contributing to metastasis.

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 approximately 1 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 peri-tumor lymphatic density and promote lymphatic invasion.

Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints.

The novel hypothesis that the secretion of the joint lubricant hyaluronan (HA) is coupled to movement has implications for normal function and osteoarthritis, and was tested in the knee joints of anaesthetized rabbits. After washing out the endogenous synovial fluid HA (miscibility coefficient 0.4), secretion into the joint cavity was measured over 5 h in static joints and in passively cycled joints. The net static secretion rate (11.2 +/- 0.7 microg h(-1), mean +/- s.e.m., n = 90) correlated with the variable endogenous HA mass (mean 367 +/- 8 microg), with a normalized value of 3.4 +/- 0.2 microg h(-1) (100 microg)(-1) . Cyclic joint movement approximately doubled the net HA secretion rate to 22.6 +/- 1.2 microg h(-1) (n = 77) and raised the normalized percentage to 5.9 +/- 0.3 microg h(-1) (100 microg)(-1). Secretion was inhibited by 2-deoxyglucose and iodoacetate, confirming active secretion. The net accumulation rate underestimated true secretion rate due to some trans-synovial loss. HA turnover time (endogenous mass/secretion rate) was 17-30 h (static) to 8-15 h (moved) The results demonstrate for the first time that the active secretion of HA is coupled to joint usage. Movement-secretion coupling may protect joints against the damaging effects of repetitive joint use, replace HA lost during periods of immobility (overnight), and contribute to the clinical benefit of exercise therapy in moderate osteoarthritis.

Clinical assessment of human lymph flow using removal rate constants of interstitial macromolecules: a critical review of lymphoscintigraphy.

Edema is a common clinical problem, and the daily avoidance of edema depends critically on the lymphatic system, which clears leaked plasma proteins and fluid from the interstitial compartment. There is often confusion as to the difference between chronic edema and lymphedema. Lymphedema is by definition primarily a disease of impaired lymphatic drainage and lymph flow, and progress in lymphedema research, currently an increasingly active field, requires a clinically viable method for the quantitative assessment of lymph drainage rate in patients. Measurement of the rate of clearance of a new protein marker, radiolabelled human immunoglobulin, from skin, subcutis, and muscle provides a way of measuring human lymph flow quantitatively and is the only viable clinical method currently available. Considerable strides have been made over the last 5-10 years in evaluating the method and its pitfalls, including potential complications such as vascular clearance, peripheral lymphovenous communications and label dissociation. The review assesses critically, for the first time, the evidence relating to the method: its pitfalls; human lymph flow in various healthy and oedematous tissues; and how this is altered in hyperfiltration edemas, inflammation, vasoconstriction and various primary and secondary human lymphedemas.

Mechanosensitive synoviocytes: a Ca2+ -PKCalpha-MAP kinase pathway contributes to stretch-induced hyaluronan synthesis in vitro.

Hyaluronan (HA) is central to joint function, contributing to synovial fluid retention, lubrication, matrix organisation and joint embryogenesis. HA synthesis by intimal synoviocytes is stimulated by stretch (SSHA), linking HA production to joint usage; but the signal transduction paths are unknown. Low passage rabbit synoviocytes (RS), cultured from micro dissected synovial intima, were subjected to 10min of 10% static stretch followed by 170-min relaxation, or to sustained stretch for 180min in a Flexcell 2000 apparatus. Medium HA content was analysed by a HA-binding assay. The roles of protein kinase C (PKC) isoforms, extracellular signal-regulated kinases (ERK1/2) and Ca(2+) signalling in SSHA were tested using kinase inhibitors, Ca(2+) chelators and Ca(2+) channel activators combined with Western blots for activated kinases. Stretch increased HA secretion by 57%, independently of stretch duration. PKCalpha translocated from cytosol to membrane and triggered the phosphorylation of ERK1/2. The PKC inhibitor bisindolylmaleimide (BIM) blocked both SSHA and ERK phosphorylation, as did Gö 6976, a specific inhibitor of Ca(2+)-dependent PKC. The Ca(2+) channel activator Bay K stimulated HA secretion and ERK phosphorylation. Extra- and intra-cellular Ca(2+) chelation by EGTA and BAPTA-AM (respectively) inhibited SSHA. SSHA was also blocked by the partially selective protein kinase A inhibitor, H-89. Connective tissue growth factor, CTGF, was not involved in SSHA. Thus, stimulation of synoviocyte HA secretion by static stretch is due at least in part the o activation of a Ca(2+) influx-dependent activation of the PKCalpha-MEK-ERK1/2 cascade. This is functionally important because it links joint lubrication to joint use.

Vascular endothelial growth factor-C (VEGF-C) expression in normal human tissues.

OBJECTIVE: To characterize vascular endothelial growth factor-C (VEGF-C) protein expression in normal human tissues by immunohistochemistry (IHC). VEGF-C is a growth factor for lymphatic endothelial cells. VEGF-C mRNA and protein are expressed in a variety of cancerous tissues, but the localization of VEGF-C protein in many normal human tissues has not been clearly demonstrated to date. We therefore performed an immunohistochemical survey of the distribution of intracellular VEGF-C protein in a range of normal human tissue types. METHODS: Five microm sections were cut from archived human tissues. Sections were dewaxed, rehydrated, and subjected to microwave pretreatment. They were incubated with VEGF-C antibody before detection with biotinylated secondary antibody using 'Elite' avidin-biotin enzyme complex and diaminobenzidine substrate. The primary antibody recognized the C-terminus of the VEGF-C propeptide that is cleaved before secretion and hence only cellular protein was detected. Negative controls used the same concentration of normal goat IgG. RESULTS: Staining manifested as small punctate cytoplasmic granules. Strong expression was observed in large intestine epithelium, and mammary duct epithelium, skeletal and cardiac muscle, thyroid, ovary, and the prostate. Weaker expression was also detected in the hepatocytes close to the terminal hepatic venules of the liver, vascular smooth muscle, and placenta. No expression was consistently detected in spleen or thymus. CONCLUSIONS: Intracellular VEGF-C protein is widely expressed in many normal human adult tissues. Its expression in cancer is not therefore per se indicative of a prolymphangiogenic change. To demonstrate the latter, a quantitative change in expression level is required.

Hyaluronan secretion by synoviocytes is mechanosensitive.

Hyaluronan (HA) is an essential component of synovial interstitial matrix and synovial fluid, but the link between its production and joint use is unclear. HA secretion is enhanced by joint distension in vivo, but direct proof that synoviocytes exhibit mechanosensitive HA secretion is lacking. We tested this in vitro. Primary rabbit synoviocyte (PRS) cultures from microdissected synovial intima were subjected to 180 min of maintained 10% static stretch, or to 10 min of 10% static stretch followed by 170 min relaxation, in a Flexcell 2000 apparatus. Stretch stimulated HA secretion into the medium over 3 h by 57%. Notably, a short stretch (10 min) was as effective as sustained stretch. Actinomycin D and cycloheximide abolished stretch-stimulated HA secretion and also reduced basal HA secretion rate. RT-PCR showed that HAS2 was the major hyaluronan synthase expressed, but there was no increase in HAS2 mRNA (or other isoforms) in continuously stretched cells, and only a small increase (20%) at 180 min in cells stretched for the first 10-30 min. However HAS2 transcription increased 10-fold in response to TGF-beta1 and IL-1beta. Thus HA secretion by intimal synoviocytes is regulated by a mechanosensitive pathway which depends on transcription and de novo protein synthesis, possibly of HAS2, but also of other proteins involved in HA secretion.

An investigation of lymphovenous communications in the upper limbs of breast cancer patients.

BACKGROUND: Approximately 25% of breast cancer patients who undergo treatment to the axilla develop breast cancer-related lymphoedema (BCRL). The aim of this study was to test the hypothesis that lymphovenous communications (LVCs) open and act as a protective mechanism against the development of BCRL. METHODS: Five patients (Group 1) received intradermal injections of (99m)Technetium-labelled autologous erythrocytes into the 2nd ipsilateral hand webspace before and 6-12 weeks following axillary node clearance surgery (ANC). Ten patients at least three years after ANC were also recruited (Group 2); seven had developed BCRL and three had not. Blood was sampled from ipsilateral and contralateral antecubital veins 5, 15, 30, 60, 120 and 180 min post-injection to assess pre-nodal shunting from lymph to blood (LVCs), since nodes block erythrocyte transit. The proportion of activity remaining in the depot was used to calculate the degree of shunting in those with evidence of LVCs. RESULTS: Significant erythrocyte-bound activity, increasing over time, was detected contralaterally in 3 of the 5 patients from Group 1 (none of whom developed BCRL) and 3 of 7 patients with BCRL from Group 2, which indicated the presence of LVCs. The degree of shunting was more marked in those patients who did not develop BCRL compared with those who did. CONCLUSIONS: The time-course of erythrocyte-bound contralateral activity indicates transit through lymphovenous communications rather than needle-induced trauma. Lymphovenous communications large enough to transmit erythrocytes are probably constitutional rather than induced. A larger study is warranted to assess any resulting protection against BCRL.

A constitutional predisposition to breast cancer-related lymphoedema and effect of axillary lymph node surgery on forearm muscle lymph flow.

AIM: The aims of this prospective study were (a) to examine the relationship between pre-operative muscle lymph flow and the predisposition to BCRL in women treated by axillary nodal surgery for breast cancer; and (b) to test the 'stopcock' hypothesis that axillary lymph node surgery impairs forearm lymph flow in the short term. METHODS: (99m)Tc-nanocoll was injected intramuscularly into both forearms of women undergoing surgery for breast cancer. Lymphatic clearance rate constant, k, representing lymph flow per unit interstitial fluid volume, was measured as the fractional disappearance rate of radioactivity from the depot site by gamma camera imaging. Axillary lymph node activity was calculated as percentage injected activity. BCRL was assessed by clinical examination and upper limb perometry. RESULTS: Of 38 pre-operative women, 33 attended at 8 ± 6 weeks post-operatively and 31 at 58 ± 9 weeks post-operatively. Seven patients (18%) developed BCRL. Prior to surgery the BCRL-destined patients had a higher mean k (0.0962 ± 0.034%/min) than non-BCRL patients (0.0830 ± 0.019%/min) (p = 0.10, unpaired t test). Post-operative k values were not significantly different from pre-operative, in either the ipsilateral (operated) or contralateral limb. Also, post-operative k values did not differ significantly between both upper limbs. Furthermore, there was no significant difference between pre- and post-operative axillary activity. CONCLUSION: Patients who develop BCRL have high lymph flow pre-surgery, which may predispose them to lymphatic overload and failure. Axillary lymph node surgery has no early, measurable effect on forearm muscle lymph flow despite surgical disruption of routes of lymph drainage.

Control of cutaneous blood vessels in psoriatic plaques.

The aim of this study was to compare local blood flow in psoriatic plaques before and after provocations known to alter cutaneous vascular resistance, in order to determine whether plaque hyperemia is caused by a failure of normal vascular control mechanisms. Cutaneous blood flow was recorded using a laser Doppler flowmeter over plaque skin (plaque site) and clinically normal skin (nonplaque site) on the opposite arm, at least 5 cm away from the nearest plaque. It is important to note that most of the laser Doppler signal comes from the subpapillary plexus of the skin and only a small portion (2%-10%) is produced by capillary blood flow. In the psoriatic plaques the basal flux was between nine and 13 times greater than nonplaque skin. The biologic zero (a signal independent of perfusion, which also persists after complete proximal arterial occlusion) was also significantly greater at plaque sites compared with nonplaque sites. Sympathetic and local vasoconstriction in psoriatic skin was shown to be intact and responses to vasodilator tests were likewise intact, i.e., there was no failure of response to normal vascular control mechanisms, albeit some quantitative differences. Tests of vasodilatation indicated that, although basal flux is high in plaque compared with nonplaque skin, arterioles supplying plaque skin can dilate further, i.e., lesional arterioles are not normally maximally dilated but have a basal constrictor tone. Interestingly, the red cell flux at maximum dilatation in nonplaque skin is less than even the basal flux in plaque skin. This means that in plaque skin either there are more arterioles than in nonplaque skin, or there is chronic, structural widening of the existing arterioles in plaque skin.

Asymmetrical effects of albumin on transsynovial fluid movement.

The effect of intraarticular infusions of albumin solution on transsynovial flow was studied in healthy rabbit knee joints and compared with the effect of albumin solution perfused through the synovial microcirculation. Increasing intravascular albumin levels enhanced fluid absorption from the joint cavity, whereas increasing intraarticular albumin levels reduced the absorption rate. The slope of intraarticular pressure-versus-absorption rate plots was reduced by albumin in proportion to the reduction in fluidity (1/viscosity). When joint pressure was held constant, the transsynovial absorption rate was reduced by albumin in excess of the fluidity reduction and even reversed to filtration into the joint cavity. Thus intraarticular albumin acts by a dual mechanism, namely by increasing synovial interstitial fluid viscosity and by exerting a peri-capillary oncotic pressure. However, the latter effect was much less than that of intravascular albumin. Reasons for this are discussed.

Chronic arm edema following breast cancer treatment.

Chronic edema of the arm (postmastectomy edema, PME) is a common and incurable complication of breast cancer treatment, often developing without warning months or years later. Although the original cause of PME is damage to axillary lymph drainage routes by surgery and radiotherapy, many observations suggest that additional factors are involved. Recent attention has focused on the Starling forces in the skin and subcutis in PME. An important finding was that the protein concentration, and hence colloid osmotic pressure, of the subcutaneous interstitial fluid of the PMF arm is unexpectedly lower than in the unaffected arm, correlating negatively with the degree of swelling. There are several possible explanations for this, such as a rise in capillary filtration rate, or interstitial proteolysis. A systemic component to PME is suggested by the finding of a lower plasma protein concentration in affected women compared with a matched postmastectomy group without swelling. A recent study using intra-vital capillaroscopy has indicated that angiogenesis occurs in the skin in PME, and an increased capillary surface area for filtration could result in an increased fluid load on a compromised lymph drainage system. Further elucidation of the pathophysiological processes in PME, in particular the adjustments to Starling forces, will enable more effective therapy of this distressing condition.

Impairment of lymph drainage in subfascial compartment of forearm in breast cancer-related lymphedema.

BACKGROUND: In arm lymphedema secondary to axillary surgery and radiotherapy (breast cancer-related lymphedema), the swelling is largely epifascial and lymph flow per unit epifascial volume is impaired. The subfascial muscle compartment is not measurably swollen despite the iatrogenic damage to its axillary drainage pathway, but this could be due to its low compliance. Our aim was to test the hypothesis that subfascial lymph drainage too is impaired. METHODS AND RESULTS: Quantitative lymphoscintigraphy was used to measure the removal rate constant (local lymph flow per unit distribution volume) for technetium-99m-human immunoglobulin G injected intramuscularly in the forearms of nine women with unilateral lymphedema. The removal rate constant was on average 31% lower in the ipsilateral swollen forearm than in the contralateral forearm (swollen arm: -0.096+/-0.041% min(-1), contralateral arm: -0.138+/-0.037% min(-1); mean+/-SD, p = 0.037). The decrease in subfascial rate constant correlated strongly with increase in arm volume (r = -0.88, p = 0.002), even though the swelling is mainly epifascial. There was no convincing evidence of dermal backflow. CONCLUSIONS: Lymph flow in the subfascial muscle compartment is decreased in breast cancer-related lymphedema. The correlation between impairment of subfascial drainage and epifascial arm swelling could be because both depend on the severity of axillary damage, or because loss of function in subfascial lymphatics impairs drainage from the epifascial to the subfascial system.

Quantification of rate and depth of pitting in human edema using an electronic tonometer.

An instrument (tonometer) was developed to measure objectively the rate as well as depth of pitting of edematous limbs under a sudden local load. Displacement versus time curves were obtained in vivo in postmastectomy edema arms and also in vitro (compression of sponges) and were analyzed in terms of spring and dashpot constants. There was no significant difference between the quasi-instantaneous indentation of tissue in the edematous and normal arms (median 2.9mm), and the two correlated strongly (r = 0.91, p < 0.0001). An exponentially slowing indentation followed. The mean difference between initial and final deformation (X infinity-X0) was greater in the swollen arms (5.7mm) than in the normal arms (1.3mm, p < 0.01). The time constant of indentation (tau) was significantly greater in the swollen arms (227s) than in the normal arms (71s). There was no correlation between the duration of the edema and any of the pitting characteristics. There was a significant negative correlation between glycosaminoglycan concentration of interstitial fluid and rate constant 1/tau (r = 0.9, p < 0.01). The tonometer thus provides an objective way of quantifying the rate and depth of pitting edema.

Validation of an optoelectronic limb volumeter (Perometer).

The Perometer, a device designed for the measurement of limb volume, has been rigorously assessed by comparison with other methods. Differences in the volume of geometric shapes and limbs determined by the Perometer and a tape measure/meter rule (i.e., Perometer minus direct measurement) were -0.8 to 2.4% (cylinders), -4.6% (truncated cone), -3.3% (mannequin limbs), 6.1% (normal human arms) and 6.8% (lymphedema arms). The larger differences were likely to be due to deviation from circular or elliptical cross-section (Perometer or tape method) and compression of the arm (tape method). Errors arising from incorrect positioning within the measuring frame were generally small, but large errors occurred when a cylinder was partially rotated within the frame (i.e., no longer perpendicular to the light beams). The Perometer was highly reproducible, each measurement taking only a few seconds. When recording the change in volume with time of a segment of arm during venous occlusion (blood flow measurement by venous occlusion plethysmography) using the Perometer plus a mercury strain gauge, between-method differences for individual blood flow recordings were apparent. The source of these differences is discussed. However, using the average of a number of blood flow recordings the Perometer and the strain gauge agreed fairly closely for both the normal and lymphedema arms. The Perometer is thus a reliable and convenient tool for the measurement of limb volume, and may also be used to measure the rate of swelling during venous occlusion plethysmography.

Assessment of truncal edema following breast cancer treatment using modified Harpenden skinfold calipers.

After initial treatment for breast cancer, lymphedema often affects the trunk as well as the arm. Evaluation of truncal swelling by the clinical "pinch test" of the posterior axillary fold is unreliable. Our aim was to develop an objective measurement, using modified Harpenden skinfold calipers. Standard Harpenden skinfold calipers exert a pressure of 12.6 g.mm-2, which rapidly squeezes edema fluid out of the skinfold. Springs were substituted to exert a lighter but relatively constant load (3.7 g.mm-2). Repeated skinfold thickness measurements on the same, normal subject then gave a relative standard deviation (r.s.d.) or coefficient of variation of 5%. The posterior axillary folds of 14 patients (age 56 +/- 13 (s.d.) years) with an average 30% arm swelling were measured using the same procedure. Readings were taken at 10 s, and again after 60 s of sustained application to assess the rate of creep, or deformation with time, attributed to displacement of pressurized interstitial fluid. Two patients had clinically observable axillary fold swelling. Eight patients, including the above two, showed axillary fold swelling by caliper measurement, defined as a 10% increase over the contralateral side (2 r.s.d.'s). Creep was greater on the affected side in all 14 patients. Thus, modified calipers can detect axillary fold edema, and thereby provide an objective method for assessing changes in swelling after lymphedema treatment.

Interstitial matrix proteins determine hyaluronan reflection and fluid retention in rabbit joints: effect of protease.

Hyaluronan (HA) retention inside the synovial cavity of joints serves diverse protective roles. We tested the hypothesis that HA retention is mediated by the network of extracellular matrix proteins in the synovial lining. Cannulated rabbit knee joints were infused with HA solution with or without pretreatment by chymopapain, a collagen-sparing protease. Trans-synovial fluid escape rate was measured and, after a period of trans-synovial filtration, samples of intra-articular fluid and subsynovial fluid were analysed for HA to assess its trans-synovial ultrafiltration. In control joints, HA ultrafiltration was confirmed by postfiltration increases in intra-articular HA concentration (259 +/- 17% of infused concentration) and reduced subsynovial concentration (30 +/- 8%; n = 11). The proportion of HA molecules reflected by the synovium was 57-75%. Chymopapain treatment increased the hydraulic permeability of the synovial lining approximately 13-fold, almost abolished the trans-synovial difference in HA concentration and reduced the HA reflected fraction to 3-7% (n = 6; P < 0.001, ANOVA). Structural studies confirmed that chymopapain treatment depleted the matrix of proteoglycans but preserved its collagen. The findings thus demonstrate that HA ultrafiltration and synovial hydraulic permeability are determined by the network of non-collagen, extracellular matrix proteins. This may be important clinically, since protease activity is raised in rheumatoid arthritis, as are HA and fluid escape.

Human lymphatic pumping measured in healthy and lymphoedematous arms by lymphatic congestion lymphoscintigraphy.

Axillary surgery for breast cancer partially obstructs lymph outflow from the arm, chronically raising the lymphatic smooth muscle afterload. This may lead to pump failure, as in hypertensive cardiac failure, and could explain features of breast cancer treatment-related lymphoedema (BCRL) such as its delayed onset. A new method was developed to measure human lymphatic contractility non-invasively and test the hypothesis of contractile impairment. 99mTc-human IgG (Tc-HIG), injected into the hand dermis, drained into the arm lymphatic system which was imaged using a gamma-camera. Lymph transit time from hand to axilla, ttransit, was 9.6+/-7.2 min (mean+/-s.d.) (velocity 8.9 cm min(-1)) in seven normal subjects. To assess lymphatic contractility, a sphygmomanometer cuff around the upper arm was inflated to 60 mmHg (Pcuff) before 99mTc-HIG injection and maintained for>>ttransit. When Pcuff exceeded the maximum pressure generated by the lymphatic pump (Ppump), radiolabelled lymph was held up at the distal cuff border. Pcuff was then lowered in 10 mmHg steps until 99mTc-HIG began to flow under the cuff to the axilla, indicating Ppump>or=Pcuff. In 16 normal subjects Ppump was 39+/-14 mmHg. Ppump was 38% lower in 16 women with BCRL, namely 24+/-19 mmHg (P=0.014, Student's unpaired t test), and correlated negatively with the degree of swelling (12-56%). Blood radiolabel accumulation proved an unreliable measure of lymphatic pump function. Lymphatic congestion lymphoscintigraphy thus provided a quantitative measure of human lymphatic contractility without surgical cut-down, and the results supported the hypothesis of lymphatic pump failure in BCRL.