Analysis on efficacy of magnetic resonance lymphangiography using INV-001 in healthy beagle dogs.

We aimed to conduct a proof-of-concept study of INV-001 in visualizing lymphatic vessels and nodes without venous contamination and to determine the optimal dose condition of INV-001 for magnetic resonance lymphangiography (MRL) in healthy beagles. MRL was performed using a 3.0-Tesla (T) whole body clinical magnetic resonance imaging (MRI) scanner. A dose-finding study of INV-001 for MRL in beagles (N = 6) was carried out according to an adaptive optimal dose finding design. For the reproducibility study (N = 6), MRL was conducted at selected INV-001 doses (0.056 and 0.112 mg Fe/kg) with a 15 mM concentration. Additionally, an excretion study (N = 3) of INV-001 was conducted by analyzing T1, T2, and T2* maps of the liver and kidney 48 h post-administration. INV-001 administration at doses of 0.056 and 0.112 mg Fe/kg (concentration: 15 mM) consistently demonstrated the visualization of contrast-enhanced lymphatic vessels and nodes without venous contamination in the beagles. The contrast enhancement effect was highest at 30 min after INV-001 administration, then gradually decreasing. No toxicity-related issues were identified during the study. After 48 h, the T1, T2, and T2* values in the liver and both kidneys were found to be comparable to the pre-administration values, indicating thorough INV-001 excretion. The optimal dosing conditions of INV-001 for MRL for contrast-enhanced visualization of lymphatic vessels and nodes exclusively with no venous contamination in beagles was determined to be 0.056 mg Fe/kg with a 15 mM concentration.

Hyaluronic Acid as a LYVE-1 Receptor Ligand in the Lymphatic System of Healthy Human Skin.

Immunohistochemical detection of the LYVE-1 marker in healthy human full-thickness skin (the epidermis and the dermis) was carried out. LYVE-1 expression was found in the endothelium of lymphatic capillaries located in the papillary dermis, in the endothelium of larger lymphatic vessels of the reticular dermis, and in fibroblasts, which indicates their joint participation in hyaluronan metabolism. LYVE-1+ staining detected for the first time in cells of the stratum basale, the stratum spinosum, and the stratum granulosum of healthy human epidermis indicates their participation in hyaluronan metabolism and allows us to consider the spaces between epidermis cells as prelimphatics.

Robot-assisted lymphovenous anastomosis surgery for lymphocele in the groin.

We present the first-in-human robot-assisted microsurgery on a lymphocele in the groin involving a man in his late 60s who had been coping with the condition for 12 months. Despite numerous efforts at conservative treatment and surgical intervention, the lymphocele persisted, leading to a referral to our clinic.Diagnostic techniques, including indocyanine green lymphography and ultrasound, identified one lymphatic vessel draining into the lymphocele. The surgical intervention, conducted with the assistance of a robot and facilitated by the Symani Surgical System (Medical Microinstruments, Calci, Italy), involved a lymphovenous anastomosis and excision of the lymphocele. An end-to-end anastomosis was performed between the lymphatic and venous vessels measuring 1 mm in diameter, using an Ethilon 10-0 suture.The surgery was successful, with no postoperative complications and a prompt recovery. The patient was discharged 3 days postoperatively and exhibited complete recovery at the 14-day follow-up. This case marks the first use of robot-assisted microsurgical lymphovenous anastomosis to address a groin lymphocele, highlighting the benefit of advanced robotic technology in complex lymphatic surgeries.

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Angpt1 binding to Tie1 regulates the signaling required for lymphatic vessel development in zebrafish.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.

Central conducting lymphatic anomaly: from bench to bedside.

Central conducting lymphatic anomaly (CCLA) is a complex lymphatic anomaly characterized by abnormalities of the central lymphatics and may present with nonimmune fetal hydrops, chylothorax, chylous ascites, or lymphedema. CCLA has historically been difficult to diagnose and treat; however, recent advances in imaging, such as dynamic contrast magnetic resonance lymphangiography, and in genomics, such as deep sequencing and utilization of cell-free DNA, have improved diagnosis and refined both genotype and phenotype. Furthermore, in vitro and in vivo models have confirmed genetic causes of CCLA, defined the underlying pathogenesis, and facilitated personalized medicine to improve outcomes. Basic, translational, and clinical science are essential for a bedside-to-bench and back approach for CCLA.

The Lymphatic Vascular System: Does Nonuniform Lymphangion Length Limit Flow-Rate?

A previously developed model of a lymphatic vessel as a chain of lymphangions was investigated to determine whether lymphangions of unequal length reduce pumping relative to a similar chain of equal-length ones. The model incorporates passive elastic and active contractile properties taken from ex vivo measurements, and intravascular lymphatic valves as transvalvular pressure-dependent resistances to flow with hysteresis and transmural pressure-dependent bias to the open state as observed experimentally. Coordination of lymphangion contractions is managed by marrying an autonomous transmural pressure-dependent pacemaker for each lymphangion with bidirectional transmission of activation signals between lymphangions, qualitatively matching empirical observations. With eight lymphangions as used here and many nonlinear constraints, the model is capable of complex outcomes. The expected flow-rate advantage conferred by longer lymphangions everywhere was confirmed. However, the anticipated advantage of uniform lymphangions over those of unequal length, compared in chains of equal overall length, was not found. A wide variety of dynamical outcomes was observed, with the most powerful determinant being the adverse pressure difference, rather than the arrangement of long and short lymphangions. This work suggests that the wide variation in lymphangion length which is commonly observed in collecting lymphatic vessels does not confer disadvantage in pumping lymph.

The use of superficial circumflex iliac perforator (SCIP) pedicle vein for lymphovenous anastomosis to treat inguinal lymphatic fistula: A case report.

The management of lymphatic fistulas following surgical procedures, in particular after inguinal lymphadenectomy, represents a significant clinical challenge. The current case report shows the novel use of the superficial circumflex iliac perforator (SCIP) pedicle vein for lymphovenous anastomosis (LVA) to treat a chronic inguinal lymphatic fistula in a 58-year-old male patient. This patient had developed a persistent lymphorrhea and wound dehiscence after a right inguinal lymph node biopsy performed for oncological reasons 1.5 months before. Pre-operative assessment with indocyanine green (ICG) lymphography confirmed a substantial lymphatic contribution to the wound discharge, thus guiding the surgical strategy. During the procedure, a pedicled tissue segment containing the SCIV was dissected and utilized to fill the wound's dead space and facilitate LVA with the leaking lymphatic vessel. Notably, a coupler device was employed for the anastomosis due to the large caliber of the lymphatic vessel involved, a technique not commonly reported in lymphatic surgeries. The result of the procedure was successful, with intra-operative ICG imaging confirming the patency of the anastomosis. After surgery the wound healed without complications. This case illustrates the potential of SCIV employment in lymphatic fistula repair in the inguinal region. While further research is needed to validate these findings, this report provides an unconventional approach to a relatively common problem in clinical practice.

Aqueous humor as eye lymph: A crossroad between venous and lymphatic system.

Aqueous humor (AQH) is a transparent fluid with characteristics similar to those of the interstitial fluid, which fills the eyeball posterior and anterior chambers and circulates in them from the sites of production to those of drainage. The AQH volume and pressure homeostasis is essential for the trophism of the ocular avascular tissues and their normal structure and function. Different AQH outflow pathways exist, including a main pathway, quite well defined anatomically and referred to as the conventional pathway, and some accessory pathways, more recently described and still not fully morphofunctionally understood, generically referred to as unconventional pathways. The conventional pathway is based on the existence of a series of conduits starting with the trabecular meshwork and Schlemm's Canal and continuing with a system of intrascleral and episcleral venules, which are tributaries to veins of the anterior segment of the eyeball. The unconventional pathways are mainly represented by the uveoscleral pathway, in which AQH flows through clefts, interstitial conduits located in the ciliary body and sclera, and then merges into the aforementioned intrascleral and episcleral venules. A further unconventional pathway, the lymphatic pathway, has been supported by the demonstration of lymphatic microvessels in the limbal sclera and, possibly, in the uvea (ciliary body, choroid) as well as by the ocular glymphatic channels, present in the neural retina and optic nerve. It follows that AQH may be drained from the eyeball through blood vessels (TM-SC pathway, US pathway) or lymphatic vessels (lymphatic pathway), and the different pathways may integrate or compensate for each other, optimizing the AQH drainage. The present review aims to define the state-of-the-art concerning the structural organization and the functional anatomy of all the AQH outflow pathways. Particular attention is paid to examining the regulatory mechanisms active in each of them. The new data on the anatomy and physiology of AQH outflow pathways is the key to understanding the pathophysiology of AQH outflow disorders and could open the way for novel approaches to their treatment.

Decorin suppresses tumor lymphangiogenesis: A mechanism to curtail cancer progression.

The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a prosurvival program and to sustain a proangiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we identified that decorin down-regulated a cluster of tumor-associated genes involved in lymphatic vessel (LV) development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of LVs, were markedly suppressed at both the mRNA and protein levels, and this suppression correlated with a significant reduction in tumor LVs. We further identified that soluble decorin, but not its homologous proteoglycan biglycan, inhibited LV sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with vascular endothelial growth factor receptor 3 (VEGFR3), the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we identified that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a biological factor with antilymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

Role of the Lymphatics in Cardiac Disease.

Cardiovascular diseases remain the largest cause of death worldwide with recent evidence increasingly attributing the development and progression of these diseases to an exacerbated inflammatory response. As a result, significant research is now focused on modifying the immune environment to prevent the disease progression. This in turn has highlighted the lymphatic system in the pathophysiology of cardiovascular diseases owing, in part, to its established function in immune cell surveillance and trafficking. In this review, we highlight the role of the cardiac lymphatic system and its potential as an immunomodulatory therapeutic target in selected cardiovascular diseases.

Interactions between nanoparticles and lymphatic systems: Mechanisms and applications in drug delivery.

The lymphatic system has garnered significant attention in drug delivery research due to the advantages it offers, such as enhancing systemic exposure and enabling lymph node targeting for nanomedicines via the lymphatic delivery route. The journey of drug carriers involves transport from the administration site to the lymphatic vessels, traversing the lymph before entering the bloodstream or targeting specific lymph nodes. However, the anatomical and physiological barriers of the lymphatic system play a pivotal role in influencing the behavior and efficiency of carriers. To expedite research and subsequent clinical translation, this review begins by introducing the composition and classification of the lymphatic system. Subsequently, we explore the routes and mechanisms through which nanoparticles enter lymphatic vessels and lymph nodes. The review further delves into the interactions between nanomedicine and body fluids at the administration site or within lymphatic vessels. Finally, we provide a comprehensive overview of recent advancements in lymphatic delivery systems, addressing the challenges and opportunities inherent in current systems for delivering macromolecules and vaccines.

Peripheral nervous system lymphatic vessels: A simple delivery route to promote nerve regeneration.

The structural and functional features of lymphatic vessels in the peripheral nervous system (pLVs) is still unclear. Here, we clarify the existence of pLVs in rats, PROX1-EGFP transgenic mice and human, and exhibit a clear three-dimensional structure for helping understand its structural features. Moreover, two specific phenotypes of lymphatics endothelial cells (Rnd1Hi LECs and Ccl21Hi LECs) in peripheral nerves are well characterized by single-cell sequencing. Subsequently, the ability of trans-lymphatic delivery to peripheral nerves via pLVs has been dynamically demonstrated. After peripheral nerve injury (PNI), extensive lymphangiogenesis occurs in the lesion area and further enhances the efficiency of retrograde lymphatic-nerve transport. In PNI animal models, subcutaneously footpad-injected exosomes are efficiently delivered to sciatic nerve via pLVs which can promote nerve regeneration. The trans-lymphatic delivery to peripheral nerves via pLVs can subtly bypass BNB which provides an easy and alternative delivery route for PNI treatment.

Real-Time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels.

The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i). This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i and diameter measurements continuously in real time. The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.