Effect of belimumab on kidney-related outcomes in patients with lupus nephritis: post hoc subgroup analyses of the phase 3 BLISS-LN trial.

BACKGROUND: Data on belimumab efficacy in patients with lupus nephritis (LN) according to diagnosis duration or induction therapy are limited. Post hoc analyses of the phase 3, randomized, double-blind BLISS-LN study (GSK BEL114054; NCT01639339) were performed to assess belimumab efficacy on kidney-related outcomes in newly diagnosed and relapsed LN subgroups and according to the use of glucocorticoid (GC) pulses at induction. METHODS: BLISS-LN randomized 448 patients with active LN to monthly intravenous belimumab 10 mg/kg or placebo plus standard therapy. Post hoc analyses assessed primary efficacy renal response (PERR) and complete renal response (CRR) at week 104, time to kidney-related event or death and time to first LN flare from week 24 in newly diagnosed and relapsed patients and patients with/without GC pulses at induction. RESULTS: A greater proportion of patients achieved a PERR with belimumab versus placebo in the newly diagnosed {69/148 [46.6%] versus 55/148 [37.2%]; odds ratio [OR] 1.36 [95% confidence interval (CI) 0.85-2.20]} and relapsed [27/75 (36.0%) versus 17/75 (22.7%); OR 2.31 (95% CI 1.07-5.01)] subgroups. Similarly for CRR [newly diagnosed: 50/148 (33.8%) versus 36/148 (24.3%); OR 1.49 (95% CI 0.88-2.51) and relapsed: 17/75 (22.7%) versus 8/75 (10.7%); OR 3.11 (95% CI 1.16-8.31)]. The probability of kidney-related event or death, or LN flare was lower with belimumab versus placebo in both subgroups. Belimumab was associated with improved kidney outcomes versus placebo with or without GC pulses at induction. CONCLUSION: Data suggest consistent benefits of belimumab on kidney outcomes for newly diagnosed and relapsed patients, and irrespective of GC pulses at induction.

TRPV4 channels' dominant role in the temperature modulation of intrinsic contractility and lymph flow of rat diaphragmatic lymphatics.

The lymphatic system drains and propels lymph by extrinsic and intrinsic mechanisms. Intrinsic propulsion depends upon spontaneous rhythmic contractions of lymphatic muscles in the vessel walls and is critically affected by changes in the surrounding tissue like osmolarity and temperature. Lymphatics of the diaphragm display a steep change in contraction frequency in response to changes in temperature, and this, in turn, affects lymph flow. In the present work, we demonstrated in an ex vivo diaphragmatic tissue rat model that diaphragmatic lymphatics express transient receptor potential channels of the vanilloid 4 subfamily (TRPV4) and that their blockade by both the nonselective antagonist Ruthenium Red and the selective antagonist HC-067047 abolished the response of lymphatics to temperature changes. Moreover, the selective activation of TRPV4 channels by means of GSK1016790A mirrored the behavior of vessels exposed to increasing temperatures, pointing out the critical role played by these channels in sensing the temperature of the lymphatic vessels' environment and thus inducing a change in contraction frequency and lymph flow.NEW & NOTEWORTHY The present work addresses the putative receptor system that enables diaphragmatic lymphatics to change intrinsic contraction frequency and thus lymph flow according to the changes in temperature of the surrounding environment, showing that this role can be sustained by TRPV4 channels alone.

Nitric oxide regulates homeoprotein OTX1 and OTX2 expression in the rat myenteric plexus after intestinal ischemia-reperfusion injury.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.

Temperature-dependent modulation of regional lymphatic contraction frequency and flow.

Lymph drainage and propulsion are sustained by an extrinsic mechanism, based on mechanical forces acting from the surrounding tissues against the wall of lymphatic vessels, and by an intrinsic mechanism attributable to active spontaneous contractions of the lymphatic vessel muscle. Despite being heterogeneous, the mechanisms underlying the generation of spontaneous contractions share a common biochemical nature and are thus modulated by temperature. In this study, we challenged excised tissues from rat diaphragm and hindpaw, endowed with spontaneously contracting lymphatic vessels, to temperatures from 24°C (hindpaw) or 33°C (diaphragmatic vessels) to 40°C while measuring lymphatic contraction frequency (fc) and amplitude. Both vessel populations displayed a sigmoidal relationship between fc and temperature, each centered around the average temperature of surrounding tissue (36.7 diaphragmatic and 32.1 hindpaw lymphatics). Although the slope factor of the sigmoidal fit to the fc change of hindpaw vessels was 2.3°C·cycles-1·min-1, a value within the normal range displayed by simple biochemical reactions, the slope factor of the diaphragmatic lymphatics was 0.62°C·cycles-1·min-1, suggesting the added involvement of temperature-sensing mechanisms. Lymph flow calculated as a function of temperature confirmed the relationship observed on fc data alone and showed that none of the two lymphatic vessel populations would be able to adapt to the optimal working temperature of the other tissue district. This poses a novel question whether lymphatic vessels might not adapt their function to accommodate the change if exposed to a surrounding temperature, which is different from their normal condition.NEW & NOTEWORTHY This study demonstrates to what extent lymphatic vessel intrinsic contractility and lymph flow are modulated by temperature and that this modulation is dependent on the body district that the vessels belong to, suggesting a possible functional misbehavior should lymphatic vessels be exposed to a chronically different temperature.

Lymph flow pattern in pleural diaphragmatic lymphatics during intrinsic and extrinsic isotonic contraction.

Peripheral rat diaphragmatic lymphatic vessels, endowed with intrinsic spontaneous contractility, were in vivo filled with fluorescent dextrans and microspheres and subsequently studied ex vivo in excised diaphragmatic samples. Changes in diameter and lymph velocity were detected, in a vessel segment, during spontaneous lymphatic smooth muscle contraction and upon activation, through electrical whole-field stimulation, of diaphragmatic skeletal muscle fibers. During intrinsic contraction lymph flowed both forward and backward, with a net forward propulsion of 14.1 ± 2.9 µm at an average net forward speed of 18.0 ± 3.6 µm/s. Each skeletal muscle contraction sustained a net forward-lymph displacement of 441.9 ± 159.2 µm at an average velocity of 339.9 ± 122.7 µm/s, values significantly higher than those documented during spontaneous contraction. The flow velocity profile was parabolic during both spontaneous and skeletal muscle contraction, and the shear stress calculated at the vessel wall at the highest instantaneous velocity never exceeded 0.25 dyne/cm(2). Therefore, we propose that the synchronous contraction of diaphragmatic skeletal muscle fibers recruited at every inspiratory act dramatically enhances diaphragmatic lymph propulsion, whereas the spontaneous lymphatic contractility might, at least in the diaphragm, be essential in organizing the pattern of flow redistribution within the diaphragmatic lymphatic circuit. Moreover, the very low shear stress values observed in diaphragmatic lymphatics suggest that, in contrast with other contractile lymphatic networks, a likely interplay between intrinsic and extrinsic mechanisms be based on a mechanical and/or electrical connection rather than on nitric oxide release.

Hyperpolarization-activated cyclic nucleotide-gated channels in peripheral diaphragmatic lymphatics.

Diaphragmatic lymphatic function is mainly sustained by pressure changes in the tissue and serosal cavities during cardiorespiratory cycles. The most peripheral diaphragmatic lymphatics are equipped with muscle cells (LMCs), which exhibit spontaneous contraction, whose molecular machinery is still undetermined. Hypothesizing that spontaneous contraction might involve hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in lymphatic LMCs, diaphragmatic specimens, including spontaneously contracting lymphatics, were excised from 33 anesthetized rats, moved to a perfusion chamber containing HEPES-Tyrode's solution, and treated with HCN channels inhibitors cesium chloride (CsCl), ivabradine, and ZD-7288. Compared with control, exposure to 10 mM CsCl reduced (-65%, n = 13, P < 0.01) the contraction frequency (FL) and increased end-diastolic diameter (DL-d, +7.3%, P < 0.01) without changes in end-systolic diameter (DL-s). Ivabradine (300 µM) abolished contraction and increased DL-d (-14%, n = 10, P < 0.01) or caused an incomplete inhibition of FL (n = 3, P < 0.01), leaving DL-d and DL-s unaltered. ZD-7288 (200 µM) completely (n = 12, P < 0.01) abolished FL, while DL-d decreased to 90.9 ± 2.7% of control. HCN gene expression and immunostaining confirmed the presence of HCN1-4 channel isoforms, likely arranged in different configurations, in LMCs. Hence, all together, data suggest that HCN channels might play an important role in affecting contraction frequency of LMCs.

Diaphragmatic lymphatic vessel behavior during local skeletal muscle contraction.

The mechanism through which the stresses developed in the diaphragmatic tissue during skeletal muscle contraction sustain local lymphatic function was studied in 10 deeply anesthetized, tracheotomized adult Wistar rats whose diaphragm was exposed after thoracotomy. To evaluate the direct effect of skeletal muscle contraction on the hydraulic intraluminal lymphatic pressures (Plymph) and lymphatic vessel geometry, the maximal contraction of diaphragmatic fibers adjacent to a lymphatic vessel was elicited by injection of 9.2 nl of 1 M KCl solution among diaphragmatic fibers while Plymph was recorded through micropuncture and vessel geometry via stereomicroscopy video recording. In lymphatics oriented perpendicularly to the longitudinal axis of muscle fibers and located at <300 µm from KCl injection, vessel diameter at maximal skeletal muscle contraction (Dmc) decreased to 61.3 ± 1.4% of the precontraction value [resting diameter (Drest)]; however, if injection was at >900 µm from the vessel, Dmc enlarged to 131.1 ± 2.3% of Drest. In vessels parallel to muscle fibers, Dmc increased to 122.8 ± 2.9% of Drest. During contraction, Plymph decreased as much as 22.5 ± 2.6 cmH2O in all submesothelial superficial vessels, whereas it increased by 10.7 ± 5.1 cmH2O in deeper vessels running perpendicular to contracting muscle fibers. Hence, the three-dimensional arrangement of the diaphragmatic lymphatic network seems to be finalized to efficiently exploit the stresses exerted by muscle fibers during the contracting inspiratory phase to promote lymph formation in superficial submesothelial lymphatics and its further propulsion in deeper intramuscular vessels.

Regional lung tissue changes with mechanical ventilation and fluid load.

PURPOSE: To investigate the regional gravity-dependent impact of mechanical ventilation and fluid overload on lung extracellular matrix (ECM) in healthy lungs. MATERIALS AND METHODS: The glycosaminoglycans (GAGs) composition of the ventral and dorsal lung parenchyma was determined in anesthetized supine healthy rats mechanically ventilated for 4 hours in air: (a) at low (∼7.5 mL/kg) or high (∼ 23 mL /kg) tidal volume (V(T)) and 0 cmH2O positive end-expiratory pressure (PEEP); (b) at low or high V(T) at 5 cmH2O PEEP and (c) with or without 7 mL /(kg·h) intravenous saline infusion. RESULTS: Mechanical ventilation degraded lung ECM, with alveolar septa thinning and structural GAGs disorganization. Low V(T) ventilation was associated with significant tissue structure changes in both ventral and dorsal lung regions, while high VT mainly affected the dependent ones. PEEP decreased ECM injury mainly in the ventral lung regions, although it did not prevent matrix fragmentation and washout at high V(T). Intravascular fluid load increased lung damage prevalently in the ventral lung regions. CONCLUSION: Mechanical ventilation and fluid load may cause additive injuries in healthy lungs, mainly in ventral regions.

Spontaneous activity in peripheral diaphragmatic lymphatic loops.

The spontaneous contractility of FITC-dextran-filled lymphatics at the periphery of the pleural diaphragm was documented for the first time "in vivo" in anesthetized Wistar rats. We found that lymphatic segments could be divided into four phenotypes: 1) active, displaying rhythmic spontaneous contractions (51.8% of 197 analyzed sites); 2) stretch-activated, whose contraction was triggered by passive distension of the vessel lumen (4.1%); 3) passive, which displayed a completely passive distension (4.5%); and 4) inert, whose diameter never changed over time (39.6%). Smooth muscle actin was detected by immunofluorescence and confocal microscopy in the vessel walls of active but also of inert sites, albeit with a very different structure within the vessel wall. Indeed, while in active segments, actin was arranged in a dense mesh completely surrounding the lumen, in inert segments actin decorated the vessels wall in sparse longitudinal strips. When located nearby along the same lymphatic loop, active, stretch-activated, and passive sites were always recruited in temporal sequence starting from the active contraction. The time delay was ∼0.35 s between active and stretch-activated and 0.54 s between stretch-activated and passive segments, promoting a uniform lymph flux of ∼150/200 pl/min. We conclude that, unlike more central diaphragmatic lymphatic vessels, loops located at the extreme diaphragmatic periphery do require an intrinsic pumping mechanism to propel lymph centripetally, and that such an active lymph propulsion is attained by means of a complex interplay among sites whose properties differ but are indeed able to organize lymph flux in an ordered fashion.

Secondary Lymphedema: Clinical Interdisciplinary Tricks to Overcome an Intriguing Disease.

Secondary lymphedema is a complex pathology which is very impairing to the patient, consisting of fluid accumulation in the tissue, accompanied by alteration of the interstitial fibrous tissue matrix, deposition of cellular debris and local inflammation. It develops mostly in limbs and/or external genitals because of demolishing oncological surgery with excision of local lymph nodes, or it may depend upon inflammatory or infective diseases, trauma, or congenital vascular malformation. Its treatment foresees various approaches, from simple postural attitude to physical therapy, to minimally invasive lymphatic microsurgery. This review focuses on the different types of evolving peripheral lymphedema and describes potential solutions to single objective symptoms. Particular attention is paid to the newest lymphatic microsurgical approaches, such as lymphatic grafting and lympho-venous shunt application, to successfully heal, in the long term, serious cases of secondary lymphedema of limbs or external genitals. The presented data also emphasize the potential role of minimally invasive microsurgery in enhancing the development of newly formed lymphatic meshes, focusing on the need for further accurate research in the development of microsurgical approaches to the lymphatic vascular system.

Fluid Osmolarity Modulates the Rate of Spontaneous Contraction of Lymphatic Vessels and Lymph Flow by Means of a Cooperation between TRPV and VRAC Channels.

Lymphatic vessels are capable of sustaining lymph formation and propulsion via an intrinsic mechanism based on the spontaneous contraction of the lymphatic muscle in the wall of lymphatic collectors. Exposure to a hyper- or hypo-osmolar environment can deeply affect the intrinsic contraction rate and therefore alter lymph flow. In this work, we aimed at defining the putative receptors underlying such a response. Functional experiments were conducted in ex vivo rat diaphragmatic specimens containing spontaneously contracting lymphatic vessels that were exposed to either hyper- or hypo-osmolar solutions. Lymphatics were challenged with blockers to TRPV4, TRPV1, and VRAC channels, known to respond to changes in osmolarity and/or cell swelling and expressed by lymphatic vessels. Results show that the normal response to a hyperosmolar environment is a steady decrease in the contraction rate and lymph flow and can be prevented by blocking TRPV1 channels with capsazepine. The response to a hyposmolar environment consists of an early phase of an increase in the contraction rate, followed by a decrease. The early phase is abolished by blocking VRACs with DCPIB, while blocking TRPV4 mainly resulted in a delay of the early response. Overall, our data suggest that the cooperation of the three channels can shape the response of lymphatic vessels in terms of contraction frequency and lymph flow, with a prominent role of TRPV1 and VRACs.

Morphological, Mechanical and Hydrodynamic Aspects of Diaphragmatic Lymphatics.

The diaphragmatic lymphatic vascular network has unique anatomical characteristics. Studying the morphology and distribution of the lymphatic network in the mouse diaphragm by fluorescence-immunohistochemistry using LYVE-1 (a lymphatic endothelial marker) revealed LYVE1+ structures on both sides of the diaphragm-both in its the muscular and tendinous portion, but with different vessel density and configurations. On the pleural side, most LYVE1+ configurations are vessel-like with scanty stomata, while the peritoneal side is characterized by abundant LYVE1+ flattened lacy-ladder shaped structures with several stomata-like pores, particularly in the muscular portion. Such a complex, three-dimensional organization is enriched, at the peripheral rim of the muscular diaphragm, with spontaneously contracting lymphatic vessel segments able to prompt contractile waves to adjacent collecting lymphatics. This review aims at describing how the external tissue forces developing in the diaphragm, along with cyclic cardiogenic and respiratory swings, interplay with the spontaneous contraction of lymphatic vessel segments at the peripheral diaphragmatic rim to simultaneously set and modulate lymph flow from the pleural and peritoneal cavities. These details may provide useful in understanding the role of diaphragmatic lymphatics not only in physiological but, more so, in pathophysiological circumstances such as in dialysis, metastasis or infection.

Draining the Pleural Space: Lymphatic Vessels Facing the Most Challenging Task.

Lymphatic vessels exploit the mechanical stresses of their surroundings together with intrinsic rhythmic contractions to drain lymph from interstitial spaces and serosal cavities to eventually empty into the blood venous stream. This task is more difficult when the liquid to be drained has a very subatmospheric pressure, as it occurs in the pleural cavity. This peculiar space must maintain a very low fluid volume at negative hydraulic pressure in order to guarantee a proper mechanical coupling between the chest wall and lungs. To better understand the potential for liquid drainage, the key parameter to be considered is the difference in hydraulic pressure between the pleural space and the lymphatic lumen. In this review we collected old and new findings from in vivo direct measurements of hydraulic pressures in anaesthetized animals with the aim to better frame the complex physiology of diaphragmatic and intercostal lymphatics which drain liquid from the pleural cavity.

Lymphatic anatomy and biomechanics.

Lymph formation is driven by hydraulic pressure gradients developing between the interstitial tissue and the lumen of initial lymphatics. While in vessels equipped with lymphatic smooth muscle cells these gradients are determined by well-synchronized spontaneous contractions of vessel segments, initial lymphatics devoid of smooth muscles rely on tissue motion to form lymph and propel it along the network. Lymphatics supplying highly moving tissues, such as skeletal muscle, diaphragm or thoracic tissues, undergo cyclic compression and expansion of their lumen imposed by local stresses arising in the tissue as a consequence of cardiac and respiratory activities. Active muscle contraction and not passive tissue displacement is required to support an efficient lymphatic drainage, as suggested by the fact that the respiratory activity promotes lymph formation during spontaneous, but not mechanical ventilation. The mechanical properties of the lymphatic wall and of the surrounding tissue also play an important role in lymphatic function. Modelling of stress distribution in the lymphatic wall suggests that compliant vessels behave as reservoirs accommodating absorbed interstitial fluid, while lymphatics with stiffer walls, taking advantage of a more efficient transmission of tissue stresses to the lymphatic lumen, propel fluid through the lumen of the lymphatic circuit.

Interplay between Gut Lymphatic Vessels and Microbiota.

Lymphatic vessels play a distinctive role in draining fluid, molecules and even cells from interstitial and serosal spaces back to the blood circulation. Lymph vessels of the gut, and especially those located in the villi (called lacteals), not only serve this primary function, but are also responsible for the transport of lipid moieties absorbed by the intestinal mucosa and serve as a second line of defence against possible bacterial infections. Here, we briefly review the current knowledge of the general mechanisms allowing lymph drainage and propulsion and will focus on the most recent findings on the mutual relationship between lacteals and intestinal microbiota.

Tissue contribution to the mechanical features of diaphragmatic initial lymphatics.

The role of the mechanical properties of the initial lymphatic wall and of the surrounding tissue in supporting lymph formation and/or progression was studied in six anaesthetized, neuromuscularly blocked and mechanically ventilated rats. After mid-sternal thoracotomy, submesothelial initial lymphatics were identified on the pleural diaphragmatic surface through stereomicroscopy. An 'in vivo' lymphatic segment was prepared by securing two surgical threads around the vessel at a distance of ∼2.5 mm leaving the vessel in place. Two glass micropipettes were inserted into the lumen, one for intraluminar injections of 4.6 nl saline boluses and one for hydraulic pressure (Plymph) recording. The compliance of the vessel wall (Clymph) was calculated as the slope of the plot describing the change in segment volume as a function of the post-injection Plymph changes. Two superficial lymphatic vessel populations with a significantly different Clymph (6.7 ± 1.6 and 1.5 ± 0.4 nl mmHg−1 (mean ± S.E.M.), P < 0.001) were identified. In seven additional rats, the average elastic modulus of diaphragmatic tissue strips was determined by uniaxial tension tests to be 1.7 ± 0.3 MPa. Clymph calculated for an initial lymphatic completely surrounded by isotropic tissue was 0.068 nl mmHg−1, i.e. two orders of magnitude lower than in submesothelial lymphatics. Modelling of stress distribution in the lymphatic wall suggests that compliant vessels may act as reservoirs accommodating large absorbed fluid volumes, while lymphatics with stiffer walls serve to propel fluid through the lumen of the lymphatic vessel by taking advantage of the more efficient mechanical transmission of tissue stresses to the lymphatic lumen.

Lymphatic Vessels and Their Surroundings: How Local Physical Factors Affect Lymph Flow.

Lymphatic vessels drain and propel lymph by exploiting external forces that surrounding tissues exert upon vessel walls (extrinsic mechanism) and by using active, rhythmic contractions of lymphatic muscle cells embedded in the vessel wall of collecting lymphatics (intrinsic mechanism). The latter mechanism is the major source of the hydraulic pressure gradient where scant extrinsic forces are generated in the microenvironment surrounding lymphatic vessels. It is mainly involved in generating pressure gradients between the interstitial spaces and the vessel lumen and between adjacent lymphatic vessels segments. Intrinsic pumping can very rapidly adapt to ambient physical stimuli such as hydraulic pressure, lymph flow-derived shear stress, fluid osmolarity, and temperature. This adaptation induces a variable lymph flow, which can precisely follow the local tissue state in terms of fluid and solutes removal. Several cellular systems are known to be sensitive to osmolarity, temperature, stretch, and shear stress, and some of them have been found either in lymphatic endothelial cells or lymphatic muscle. In this review, we will focus on how known physical stimuli affect intrinsic contractility and thus lymph flow and describe the most likely cellular mechanisms that mediate this phenomenon.

Acute Exposure of Collecting Lymphatic Vessels to Low-Density Lipoproteins Increases Both Contraction Frequency and Lymph Flow: An In Vivo Mechanical Insight.

Background: Lymphatic vessels drain fluids and solutes from interstitial spaces and serosal cavities. Among the solutes, low-density lipoproteins (LDL) are drained and can be detected in peripheral lymph, where they have been reported to exert a modulatory action on lymphatic vessels intrinsic contraction rate. In the present work, we investigated lymphatic vessel mechanical properties (contraction frequency and amplitude) that may be modulated by LDL application and the consequence on lymph flow. Methods and Results: Human-derived LDL were resuspended in phosphate-buffered saline (PBS) and microinjected in the interstitial space surrounding spontaneously contracting lymphatic vessels of the rat diaphragm, in vivo. Vessels' contraction rate and diameter were measured in control conditions (PBS) and after LDL injection. Lymph flow (Jlymph) was computed from contraction rate and diameter change. In some animals, after the recording procedure, diaphragmatic tissue samples were excised and immunostained with antilymphatic muscle (LM) actin to investigate the correlation between LM signal level and contraction amplitude. Data indicate a positive, saturating correlation between the abundance of LM actin and contraction amplitude, and LDL microinjection caused an acute increase in contraction frequency (+126%), a reduction of contraction amplitude to 75% of that obtained after PBS injection, and a +63% increase in Jlymph. Conclusions: From our in vivo analysis of the mechanical parameters affected by LDL, Jlymph was increased by a predominant effect on the contraction rate rather than amplitude, suggesting that the still elusive messaging system might be linked to the pacemaker sites.

Investigation on the role of biallelic variants in VEGF-C found in a patient affected by Milroy-like lymphedema.

BACKGROUND: Milroy-like disease is the diagnostic definition used for patients with phenotypes that resemble classic Milroy disease (MD) but are negative to genetic testing for FLT4. In this study, we aimed at performing a genetic characterization and biochemical analysis of VEGF-C variations found in a female proband born with congenital edema consistent with Milroy-like disease. METHODS: The proband underwent next-generation sequencing-based genetic testing for a panel of genes associated with known forms of hereditary lymphedema. Segregation analysis was performed on family members by direct sequencing. In vitro studies were performed to evaluate the role of a novel identified variant. RESULTS: Two VEGF-C variations were found in the proband, a novel p.(Ser65Arg) and a pathogenic c.148-3_148-2delCA, of paternal and maternal origin, respectively. Functional characterization of the p.(Ser65Arg) variation in vitro showed alterations in VEGF-C processing. CONCLUSIONS: Our findings reveal an interesting case in which biallelic variants in VEGF-C are found in a patient with Milroy-like lymphedema. These data expand our understanding of the etiology of congenital Milroy-like lymphedema.

Gait screening of a population of young, healthy athletes by means of a portable, low-cost device unveils hidden left-right asymmetries in both quadriceps and anterior cruciate ligament forces.

OBJECTIVE: The present study reports the on-field screening of a population of young soccer players in the pursuit of alterations in gait using a portable and low-cost gait analysis system composed of a Wii Balance Board and a webcam. RESULTS: Recordings of motion of the lower extremities along with vertical ground reaction force (GRF) were used to quantify coefficients of symmetry for the overall GRF and the forces exerted by the quadriceps femori and acting on the anterior cruciate ligament (ACL). Data show that, in face of a quite homogeneous symmetry of GRF during left and right stance phases of gait, quadriceps and ACL exert and are subjected to left-right asymmetrical forces that might prelude, especially in young athletes, later alterations of gait.