Lymphatic Regeneration Research Articles

Improved lymphangiogenesis around vascularized lymph node flaps by periodic injection of hyaluronidase in a rodent model

Vascularized lymph node transfer (VLNT) is an advanced surgical approach for secondary lymphedema (SLE) treatment, but tissue fibrosis around the lymph node flap (VLNF) inhibiting lymphangiogenesis is the biggest challenge undermining its therapeutic efficacy. This study explored the effects of periodic hyaluronidase (HLD) injection in reducing fibrosis and promoting lymphangiogenesis in 52 Sprague–Dawley rats with a VLNF over 13 weeks. The results demonstrated that HLD administration significantly enhanced swelling reduction, lymphatic drainage efficiency, and lymphatic vessel regeneration, with up to a 26% decrease in tissue fibrosis around the VLNF. These findings suggest that combining VLNT with periodic injections of HLD could substantially improve SLE treatment outcomes in clinical settings. It offers a promising direction for future therapeutic strategies and drug development aimed at increasing the efficacy of surgical treatment for SLE patients.

Matrix Stiffness of GelMA Hydrogels Regulates Lymphatic Endothelial Cells toward Enhanced Lymphangiogenesis.

Lymphatic vessel regeneration is crucial for various tissue engineering strategies, particularly in resolving inflammation and restoring tissue homeostasis. In our study, we focused on investigating how hydrogel matrix stiffness influences lymphatic endothelial cells (LECs) in promoting lymphatic vessel regeneration. Gelatin methacrylate (GelMA) was chosen as our biomaterial due to its versatility in tissue engineering and biofabrication. We fabricated GelMA hydrogels at concentrations of 5, 7.5, and 15% (w/v) with corresponding Young's modulus values of 1.55 kPa (soft matrix), 12.02 kPa (medium matrix), and 48.50 kPa (stiff matrix). Among these, the 7.5% GelMA hydrogel exhibited optimal stiffness for promoting lymphangiogenesis. LECs seeded either on the hydrogel surface or within spontaneously formed a more stable lymphatic capillary network compared with other GelMA formulations. Furthermore, we investigated the enhancement of lymphangiogenesis by incorporating VEGF-C into the GelMA hydrogel, leveraging the synergistic effects of mechanical and chemical cues. Our results underscored the critical role of FAK-phosphorylation in this process; treatment with an FAK-specific inhibitor prevented the formation of tube-like structures by LECs and attenuated the expression of lymphatic markers. Overall, our findings highlight how the mechanical and chemical cues provided by GelMA hydrogels can effectively regulate LEC behavior toward enhanced lymphangiogenesis via the integrin/FAK mechanotransduction pathway. This study proposes a promising strategy for developing hydrogel-based scaffolds or bioinks tailored to promote lymphatic vessel regeneration in therapeutic applications.

Robust Differentiation of Human Pluripotent Stem Cells into Lymphatic Endothelial Cells Using Transcription Factors

Introduction: Generating new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Yet, compared to the blood vascular system, protocols to differentiate human induced pluripotent stem cells (hiPSCs) into lymphatic endothelial cells (LECs) are still lacking. Methods: Transcription factors, ETS2 and ETV2 are key regulators of embryonic vascular development, including lymphatic specification. While ETV2 has been shown to efficiently generate blood endothelial cells, little is known about ETS2 and its role in lymphatic differentiation. Here, we describe a method for rapid and efficient generation of LECs using transcription factors, ETS2 and ETV2. Results: This approach reproducibly differentiates four diverse hiPSCs into LECs with exceedingly high efficiency. Timely activation of ETS2 was critical, to enable its interaction with Prox1, a master lymphatic regulator. Differentiated LECs express key lymphatic markers, VEGFR3, LYVE-1, and Podoplanin, in comparable levels to mature LECs. The differentiated LECs are able to assemble into stable lymphatic vascular networks in vitro, and secrete key lymphangiocrine, reelin. Conclusion: Overall, our protocol has broad applications for basic study of lymphatic biology, as well as toward various approaches in lymphatic regeneration and personalized medicine.

ERG Regulates Lymphatic Vessel Specification Genes and Its Deficiency Impairs Wound Healing-Associated Lymphangiogenesis.

Rarefaction of blood and lymphatic vessels in the skin has been reported in systemic sclerosis (SSc) (scleroderma). E26 transformation-specific-related factor (ERG) and Friend leukemia virus-induced erythroleukemia 1 (FLI-1) are important regulators of angiogenesis, but their role in lymphatic vasculature is lesser known. The goal of this study was to determine the role of ERG and FLI-1 in postnatal lymphangiogenesis and SSc lymphatic system defects. Immunofluorescence was used to detect ERG and FLI-1 in skin biopsy samples from patients with SSc and healthy controls. Transcriptional analysis of ERG or FLI-1-silenced human dermal lymphatic endothelial cells (LECs) was performed using microarrays. Effects of ERG and FLI-1 deficiency on in vitro tubulogenesis in human dermal LECs were examined using a Matrigel assay. ERG and FLI-1 endothelial-specific knockouts and ERG lymphatic-specific knockouts were generated to examine vessel regeneration in mice. ERG and FLI-1 protein levels were reduced in the blood and lymphatic vasculature in SSc skin biopsy samples. ERG levels were shown to regulate genes involved in lymphatic vessel specification, including vascular endothelial growth factor receptor 3/FLT-4, lymphatic vessel endothelial hyaluronan receptor 1, SOX-18, and prospero homeobox 1 (PROX-1), whereas FLI-1 enhanced the function of ERG. The ERG-FLT-4 pathway regulated in vitro tubulogenesis in human LECs. Deficiency of ERG or FLI-1 similarly impaired the function of blood vessels in mice. However, only ERG deficiency affected the regeneration of lymphatic vessels during wound healing. ERG and FLI-1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI-1 in SSc endothelial cells may contribute to the impairment of blood and lymphatic vasculature in patients with SSc.

Perturbed collagen metabolism underlies lymphatic recanalization failure in Gata2 heterozygous deficient mice.

Lymphedema has become a global health issue following the growing number of cancer surgeries. Curative or supportive therapeutics have long been awaited for this refractory condition. Transcription factor GATA2 is crucial in lymphatic development and maintenance, as GATA2 haploinsufficient disease often manifests as lymphedema. We recently demonstrated that Gata2 heterozygous deficient mice displayed delayed lymphatic recanalization upon lymph node resection. However, whether GATA2 contributes to lymphatic regeneration by functioning in the damaged lymph vessels' microenvironment remains explored. In this study, our integrated analysis demonstrated that dermal collagen fibers were more densely accumulated in the Gata2 heterozygous deficient mice. The collagen metabolism-related transcriptome was perturbed, and collagen matrix contractile activity was aberrantly increased in Gata2 heterozygous embryonic fibroblasts. Notably, soluble collagen placement ameliorated delayed lymphatic recanalization, presumably by modulating the stiffness of the extracellular matrix around the resection site of Gata2 heterozygous deficient mice. Our results provide valuable insights into mechanisms underlying GATA2-haploinsufficiency-mediated lymphedema and shed light on potential therapeutic avenues for this intractable disease.

Progenitor Cell Sources for 3D Bioprinting of Lymphatic Vessels and Potential Clinical Application.

The lymphatic system maintains tissue fluid homeostasis and it is involved in the transport of nutrients and immunosurveillance. It also plays a pivotal role in both pathological and regenerative processes. Lymphatic development in the embryo occurs by polarization and proliferation of lymphatic endothelial cells from the lymph sacs, that is, lymphangiogenesis. Alternatively, lymphvasculogenesis further contributes to the formation of lymphatic vessels. In adult tissues, lymphatic formation rarely occurs under physiological conditions, being restricted to pathological processes. In lymphvasculogenesis, progenitor cells seem to be a source of lymphatic vessels. Indeed, mesenchymal stem cells, adipose stem cells, endothelial progenitor cells, and colony-forming endothelial cells are able to promote lymphatic regeneration by different mechanisms, such as direct differentiation and paracrine effects. In this review, we summarize what is known on the diverse stem/progenitor cell niches available for the lymphatic system, emphasizing the potential that these cells hold for lymphatic tissue engineering through 3D bioprinting and their translation to clinical application.

Lymphatic Drainage-Promoting Effects by Engraftment of Artificial Lymphatic Vascular Tissue Based on Human Adipose Tissue-Derived Mesenchymal Stromal Cells in Mice

Regenerative medicine using lymphatic vascular engineering is a promising approach for treating lymphedema. However, its development lags behind that of artificial blood vascular tissue for ischemic diseases. In this study, we constructed artificial 3D lymphatic vascular tissue, termed ASCLT, by co-cultivation of ECM-nanofilm-coated human adipose tissue-derived mesenchymal stromal cells (hASCs) and human dermal lymphatic endothelial cells (HDLECs). The effect of hASCs in lymphatic vessel network formation was evaluated by comparison with the tissue based on fibroblasts, termed FbLT. Our results showed that the density of lymphatic vascular network in ASCLT was higher than that in FbLT, demonstrating a promoting effect of hASCs on lymphatic vascular formation. This result was also supported by higher levels of lymphangiogenesis-promoting factors, such as bFGF, HGF, and VEGF-A in ASCLT than in FbLT. To evaluate the therapeutic effects, FbLTs and ASCLTs were subcutaneously transplanted to mouse hindlimb lymphatic drainage interruption models by removal of popliteal and subiliac lymph nodes. Despite the restricted engraftment of lymphatic vessels, ASCLT promoted regeneration of irregular and diverse lymphatic drainage in the skin, as visualized by indocyanine green imaging. Moreover, transplantation of ASCLT to the popliteal lymph node resection area also resulted in lymphatic drainage regeneration. Histological analysis of the generated drainage visualized by FITC-dextran injection revealed that the drainage was localized in the subcutaneous area shallower than the dermal muscle. These findings demonstrate that ASCLT promotes lymphatic drainage in vivo and that hASCs can serve as an autologous source for treatment of secondary lymphedema by tissue engineering.

Lymphedema Surgical Treatment Using BioBridgeTM: A Preliminary Experience

Lymphedema treatment has traditionally been based on conservative therapies. In recent years, the introduction of two microsurgical techniques—venolymphatic anastomoses and lymph-node transplantation—have revolutionized the therapeutic approach to this disease. However, these surgical techniques only provide a partial volume reduction, and thus, they require further improvement. A new device called BioBridgeTM, an implantable biocompatible and biodegradable mesh, made up of aligned nanofibrillar collagen scaffolds that mimic the collagen matrix, could significantly improve the benefits of these surgical strategies, favoring lymphatic regeneration. In this article, we describe our experience with three patients affected by primary lymphedema, two treated with a lymph-node transplant, and one with venolymphatic anastomosis. All interventions involved the placement of nanocollagen scaffolds. With each patient, non-contrast magnetic resonance lymphography was performed for surgical planning and there was an imaging and a clinical follow-up at 1 year. All patients showed a significant reduction in the affected areas with the demonstration of new lymphatic vessels at MR imaging follow-up. This was a preliminary experience, and it showed the potential benefit of positioning nonfibrillar scaffolds in combination with other microsurgical therapies for lymphedema.

Scanning Electron Microscopy Analysis of Lymphatic Regeneration in a Secondary Lymphedema Mouse Model: A Preliminary Study

Under inflammatory conditions including lymphatic disorders, bone marrow-derived myeloid cells often express lymphatic endothelial cell (LEC) markers, and these cells are then called LEC progenitor cells, which extend lymphatic vessels by fusing with existing lymphatic vessels. However, studies on the mechanism of lymphatic regeneration using three-dimensional images of lymphatic structures are limited. In this study, scanning electron microscopy (SEM) was used to observe the three-dimensional structure of lymphangiogenesis in a mouse model of secondary lymphedema. The model was established in C57BL/6J mice via circumferential incision in the inguinal region of the left hind limb. Skin samples were obtained from the lymphedema region on days 2, 5, and 8 after surgery. To determine lymphatic vessel positions using SEM analysis, we detected anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) immunoreactivity in serial sections and overlaid them during SEM observation. On days 2 and 5, spherical cells, probably myeloid cells, were attached and fused to the LYVE-1-positive lymphatic vessel walls. On day 8, spherical cells were converted to string-shaped cells, forming a new lymphatic vessel wall resembling an intraluminal pillar. Our results showed the newly formed lymphatic vessel wall extended into the lumen, suggesting intussusceptive lymphangiogenesis.

Mechanisms regulating vascular and lymphatic regeneration in the heart after myocardial infarction.

Myocardial infarction, caused by a thrombus or coronary vascular occlusion, leads to irreversible ischaemic injury. Advances in early reperfusion strategies have significantly reduced short-term mortality after myocardial infarction. However, survivors have an increased risk of developing heart failure, which confers a high risk of death at 1 year. The capacity of the injured neonatal mammalian heart to regenerate has stimulated extensive research into whether recapitulation of developmental regeneration programmes may be beneficial in adult cardiovascular disease. Restoration of functional blood and lymphatic vascular networks in the infarct and border regions via neovascularisation and lymphangiogenesis, respectively, is a key requirement to facilitate myocardial regeneration. An improved understanding of the endogenous mechanisms regulating coronary vascular and lymphatic expansion and function in development and in adult patients after myocardial infarction may inform future therapeutic strategies and improve translation from pre-clinical studies. In this review, we explore the underpinning research and key findings in the field of cardiovascular regeneration, with a focus on neovascularisation and lymphangiogenesis, and discuss the outcomes of therapeutic strategies employed to date. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

In vivo vascularized scaffold with different shear-exposed models for lymphatic tissue regeneration

Current clinical treatments on lymphedema provide promising results, but also result in donor site morbidities. The establishment of a microenvironment optimized for lymphangiogenesis can be an alternative way to enhance lymphatic tissue formation. Hemodynamic flow stimuli have been confirmed to have an influential effect on angiogenesis in tissue engineering, but not on lymphatic vessel formation. Here, the three in vivo scaffolds generated from different blood stimuli in the subcutaneous layer, in the flow through pedicle, and in an arterio-venous (AV) loop model, were created to investigate potential of lymphangiogenesis of scaffolds containing lymphatic endothelial cells (LECs). Our results indicated that AV loop model displayed better lymphangiogenesis in comparison to the other two models with slower flow or no stimuli. Other than hemodynamic force, the supplement of LECs is required for lymphatic vessel regeneration. The in vivo scaffold generated from AV loop model provides an effective approach for engineering lymphatic tissue in the clinical treatment of lymphedema.

Synthetic hyaluronic acid coating preserves the phenotypes of lymphatic endothelial cells.

Lymphatic endothelial cells (LECs) play a critical role in the formation and maintenance of the lymphatic vasculature, which is essential for the immune system, fluid balance, and tissue repair. However, LECs are often difficult to study in vivo and in vitro models that accurately mimic their behaviors and phenotypes are limited. In particular, LECs have been shown to lose their lymphatic markers over time while being cultured in vitro, which reflect their plasticity and heterogeneity in vivo. Since LECs uniquely express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we hypothesized that surface coating with hyaluronic acid (HA) can preserve LEC phenotypes and functionalities. Dopamine conjugated hyaluronic acid (HA-DP) was synthesized with 42% degree of substitution to enable surface modification and conjugation onto standard tissue culture plates. Compared to fibronectin coating and tissue culture plate controls, surface coating with HA-DP was able to preserve lymphatic markers, such as prospero homeobox protein 1 (Prox1), podoplanin (PDPN), and LYVE-1 over several passages in vitro. LECs cultured on HA-DP expressed lower levels of focal adhesion kinase (FAK) and YAP/TAZ, which may be responsible for the maintenance of the lymphatic characteristics. Collectively, the HA-DP coating may provide a novel method for culturing human LECs in vitro toward more representative studies in basic lymphatic biology and lymphatic regeneration.

Lentiviral overexpression of VEGFC in transplanted MSCs leads to resolution of swelling in a mouse tail lymphedema model.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Despite the various physical therapy and surgical options available, most treatments are palliative and fail to address the underlying lymphatic vascular insufficiency driving lymphedema progression. Stem cell therapy provides a promising alternative in the treatment of various chronic diseases with a wide range of therapeutic effects that reduce inflammation, fibrosis, and oxidative stress, while promoting lymphatic vessel (LV) regeneration. Specifically, stem cell transplantation is suggested to promote LV restoration, rebuild lymphatic circulation, and thus potentially be utilized towards an effective lymphedema treatment. In addition to stem cells, studies have proposed the administration of vascular endothelial growth factor C (VEGFC) to promote lymphangiogenesis and decrease swelling in lymphedema. Here, we seek to combine the benefits of stem cell therapy, which provides a cellular therapeutic approach that can respond to the tissue environment, and VEGFC administration to restore lymphatic drainage. Specifically, we engineered mesenchymal stem cells (MSCs) to overexpress VEGFC using a lentiviral vector (hVEGFC MSC) and investigated their therapeutic efficacy in improving LV function and tissue swelling using near infrared (NIR) imaging, and lymphatic regeneration in a single LV ligation mouse tail lymphedema model. First, we showed that overexpression of VEGFC using lentiviral transduction led to an increase in VEGFC protein synthesis in vitro. Then, we demonstrated hVEGFC MSC administration post-injury significantly increased the lymphatic contraction frequency 14-, 21-, and 28-days post-surgery compared to the control animals (MSC administration) in vivo, while also reducing tail swelling 28-days post-surgery compared to controls. Our results suggest a therapeutic potential of hVEGFC MSC in alleviating the lymphatic dysfunction observed during lymphedema progression after secondary injury and could provide a promising approach to enhancing autologous cell therapy for treating lymphedema.

The Effect of Vascular Endothelial Growth Factor C and Adipose-Derived Stem Cells on Lymphatic Regeneration in a Rat Vascularized Lymph Node Transfer Model.

Lymphedema is a chronic condition characterized by progressive edema with complicated treatment. Recently, new treatment strategies inducing lymphangiogenesis were proposed. The aim of our study was to examine the effect of vascular endothelial growth factor C (VEGF-C) and adipose-derived stem cells (ADSCs) on lymphatic regeneration and drainage re-establishment in vascularized lymph node transfer (VLNT) model using a pedicled vascularized lymph node (VLN) groin flap. Female Lewis rats with groin VLN flaps were utilized as a lymphedema model. Group A served as the control. Group B received VEGF-C. Group C received both VEGF-C and ADSCs. Group D received ADSCs only. Lymphatic drainage re-establishment was evaluated by ultrasound-photoacoustic imaging (US-PAI) after indocyanine green (ICG) injection. The fastest regeneration of elevated flaps was observed in Groups B and C in all monitored periods. After the first month, ICG positivity was detected in 14.3% of animals in Group A, 71.43% of animals in Group B (odds ratio [OR] = 15; p = 0.048), and 83.33% in Group C (OR = 30; p = 0.027). On the contrary, the difference between control group and Group D (16.67%; p = 0.905) was statistically insignificant. Administration of VEGF-C, ADSC + VEGF-C, and ADSC led to full flap regeneration after 6 months. The control group had the lowest percentage of ICG positivity at all monitored time points. We found that the fastest regeneration occurred with the combination of the VLN flap and VEGF-C. The addition of ADSC had an insignificant effect in our study. Furthermore, we proved the feasibility of PAI as an assessment tool of the lymphatic drainage recovery in a VLNT model.

A High-Fat Diet in the Absence of Obesity Increases Lymphangiogenesis by Inducing VEGF-C in a Murine Lymphedema Model.

Many researchers have attempted to induce lymphangiogenesis for the treatment of lymphedema. However, most previous studies had limited clinical usefulness. A high-fat diet (HFD) increases serum β-hydroxybutyrate (β-OHB) levels, which can stimulate lymphangiogenesis. The authors hypothesized that an HFD will ameliorate lymphedema through enhanced lymphangiogenesis. The effects of β-OHB on the lymphangiogenic process in human dermal lymphatic endothelial cells were analyzed. A mouse tail lymphedema model was used to evaluate the effects of an HFD on lymphedema. Experimental mice were fed an HFD (45% kcal as fat, 20% as protein, and 35% as carbohydrates) for 4 weeks. Tail volume was measured using the truncated cone formula. Biopsy specimens were taken 6 weeks after surgical induction of lymphedema. In human dermal lymphatic endothelial cells, treatment with 20 mM of β-OHB increased cell viability ( P = 0.008), cell migration ( P = 0.011), tube formation ( P = 0.005), and VEGF-C mRNA and protein expression ( P < 0.001) compared with controls. HFD feeding decreased tail volume by 14.3% and fibrosis by 15.8% ( P = 0.027), and increased the lymphatic vessel density ( P = 0.022) and VEGF-C protein expression ( P = 0.005) compared with those of operated, standard chow diet-fed mice. The authors' findings demonstrated that β-OHB promoted lymphatic endothelial cell function and increased VEGF-C mRNA and protein expression. When mice with tail lymphedema were fed an HFD, volume and fibrosis of the tail decreased. Therefore, the authors' findings suggest that an HFD can be a successful novel dietary approach to treating lymphedema. Lymphatic regeneration after vascularized lymph node transfer can be augmented when a high-fat diet is used in conjunction with vascularized lymph node transfer.

The Lymphangiogenic Effect of Continuous Negative Pressure Wound Therapy in Murine Incisional Wounds

INTRODUCTION: The effects of negative pressure wound therapy (NPWT) on the vascular system are well described. Despite the significant role of the lymphatics in wound healing, the impact of NPWT on lymphangiogenesis remains to be elucidated. Here, we studied the effects of NPWT on lymphatic regeneration in a murine incisional wound model. METHODS: Thirty diabetic mice (db/db) received dorsal incisional skin wounds and were treated with occlusive covering (Control, n = 15) or continuous NPWT (24 hours × 7 days, –125 mmHg; NPWT, n = 15). The wounds were monitored for 28 days, tissue was harvested on day 10 for analysis, and Evans Blue staining of lymphatic drainage was performed at 28 days. RESULTS: NPWT was associated with increased lymphatic density, as visualized using the lymphatic-vessel specific Podoplanin (39.6 ± 19.9 vs 11.5 ± 5.9 PDPN+/HPF; p < 0.001) and LYVE-1 (24.8 ± 9.1 vs 13.7 ± 7.9 LYVE-1+/HPF; p = 0.01). Lymphatic vessel diameter was higher in the NPWT group (27.5 ± 9.0 vs 12.4 ± 2.2 μm; p < 0.001). Western blotting showed that NPWT upregulated the expression of LYVE-1 at the protein level (p = 0.03). On functional assessment of the lymphatics, the NPWT exhibited a larger area of Evans Blue positivity at all timepoints (0, 5, 10, 15, 20, 25 and 30 minutes). CONCLUSION: Our findings indicate that NPWT significantly modulates lymphatic regeneration in incisional wounds. The effects were shown to occur at the protein level, resulting in structural and functional improvement. This highlights the potential of NPWT to therapeutical stimulate lymphatic growth and drainage in wound healing or in conditions such as lymphedema.

Emerging Anti-Inflammatory Pharmacotherapy and Cell-Based Therapy for Lymphedema.

Secondary lymphedema is a common complication of lymph node dissection or radiation therapy for cancer treatment. Conventional therapies such as compression sleeve therapy, complete decongestive physiotherapy, and surgical therapies decrease edema; however, they are not curative because they cannot modulate the pathophysiology of lymphedema. Recent advances reveal that the activation and accumulation of CD4+ T cells are key in the development of lymphedema. Based on this pathophysiology, the efficacy of pharmacotherapy (tacrolimus, anti-IL-4/IL-13 antibody, or fingolimod) and cell-based therapy for lymphedema has been demonstrated in animal models and pilot studies. In addition, mesenchymal stem/stromal cells (MSCs) have attracted attention as candidates for cell-based lymphedema therapy because they improve symptoms and decrease edema volume in the long term with no serious adverse effects in pilot studies. Furthermore, MSC transplantation promotes functional lymphatic regeneration and improves the microenvironment in animal models. In this review, we focus on inflammatory cells involved in the pathogenesis of lymphedema and discuss the efficacy and challenges of pharmacotherapy and cell-based therapies for lymphedema.

QS19. Negative Pressure Wound Therapy Stimulates Lymphangiogenesis in Murine Incisional Wound Healing

Purpose: Lymphangiogenesis is central to the process of wound healing as it enables transport of immune cells to and fluid removal away from the wound area. Despite the significant function of the lymphatic system in wound healing, and the frequent clinical use of Negative Pressure Wound Therapy (NPWT) in healing wounds, the impact of mechanical force application on lymphangiogenesis remains to be elucidated. In this study we utilized a murine incisional wound model to investigate the impact of NPWT on lymphatic regeneration. Methods: A dorsal incisional skin wound was induced in 30 obese and diabetic mice (db/db) which were then treated either with occlusive covering (Control, n=15) or continuous NPWT (24 hours x 7 days, -125 mmHg; NPWT, n=15). The follow up period was 28 days with tissue harvested on day 10 for histological and molecular analysis. Lymphatic function was assessed on day 28 using Evans Blue staining. Results: Lymphatic density was significantly increased in the NPWT group, verified by probing with the lymphatic vessel specific markers LYVE-1 (25±9 vs 14±8 LYVE-1+/HPF; p=0.01) and Podoplanin (40±20 vs 12±6 PDPN+/HPF; p<0.001). The diameter of the lymphatics was increased with NPWT (28±9 vs 12±2 μm; p<0.001). LYVE-1 was upregulated at the protein level as assessed with western blotting (p=0.03). Drainage was enhanced by NPWT, with the Evans Blue positive area being larger in the NPWT group at all timepoints (0, 5, 10, 15, 20, 25 and 30 minutes). Conclusion: Application of micromechanical forces promotes lymphangiogenesis in incisional wounds. The mechanism is likely multifactorial, with a direct impact on the lymphatic vessels through upregulation of LYVE-1 at the molecular level, and edema-dependent effects on the interstitial hydrostatic and oncotic pressure. Overall, NPWT can be used to stimulate lymphatic growth and function in wound healing or as a therapy for conditions such as lymphedema.

30. The Effect of Lymphatic Proliferation in Extremity Trauma And Aberrant Wound Healing

Purpose: Traumatic injury results in alterations to tissue organization requiring a highly orchestrated process for proper healing. Following traumatic injury, muscle, tendon, vasculature, nerves, and bone are destroyed resulting in impaired regeneration. There is a paucity of studies investigating the role of lymphatic vasculature network plays in tissue healing following traumatic injury. This is surprising considering the role that the lymphatic system plays in the normal inflammatory response and managing resulting edema which is critical in preventing infection and initiating the healing cascade. Alterations in this process can result in aberrant healing seen following traumatic injury. Utilizing a reliable and reproducible method of traumatic injury, we investigated the role of lymphatic vessels in and aberrant wound healing utilizing novel bioinformatic techniques and 2- and 3-dimensional confocal imaging modalities. Methods: We utilized a murine model of traumatic injury resulting in formation of heterotopic ossification (HO). We performed a 30% total body surface area burn to the dorsum of the mouse and a concurrent Achilles’ tenotomy (Burn/tenotomy; BT) resulting in fully formed HO at the Achilles’ injury site nine weeks following injury. C57B6 and Prox1-eGFP mice labeling lymphatic endothelial cells were injured. To visualize lymphatic endothelium prior to injury, uninjured hindlimbs were harvested and processed utilizing the PEGASOS method for tissue clearing. Hindlimbs were harvested prior to injury, one, three, and nine weeks post-injury for histology. Tissue was also harvested from uninjured mice, one, and six weeks post-injury for single-cell RNA-sequencing (scRNA). Separately, we performed the BT injury in chylous ascites (Chy) mice characterized by dysfunctional lymphatic system due to an inactivating mutation in Vegfr3, a cell-surface receptor critical for the growth and survival of lymphatic endothelium. Mice were harvested nine weeks post-injury to assess the impact on HO formation. Results: Reconstructed 3-D confocal microscopy images of cleared uninjured whole-mount hindlimb tissue stained for lymphatic endothelial cell markers CD31 and LYVE1 show lymphatic vessels at the injury site. Sections from uninjured Achilles’ tendon and one, three, and nine weeks post-injury from Prox1-eGFP mice co-stained with LYVE1 show lymphatics in tendon peritenon, however, no lymphatic vessels within the tendon prior to injury. Following injury, lymphatics endothelium is disrupted, characterized by no positive Prox1-eGFP staining following injury. Interestingly, Prox1-eGFP+ cells are found within the Achilles’ tendon and Prox1-eGFP/LYVE1 double-positive cells forming a lumen are found nine weeks post-injury within the Achilles’ tendon (B; n=3/group, p*<0.5). ScRNA shows expression of lymphatic endothelial growth factor Vegfc following injury by mesenchymal progenitor cells (MPCs), while receptors for the ligand are expressed by the lymphatic endothelial cell cluster. VEGFR3 mutation leads to increased variability of HO formation following injury (E; n=4/6/group). Conclusion: These findings highlight the disruption and regeneration of lymphatic vessels following extremity trauma. For the first time, we propose a mechanism for MPC-lymphatics interaction that drives lymphatic regeneration following extremity injury through VEGFC-VEGFR3 driven lymphangiogenesis.

Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymphatic Expansion.

Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.