Lymphatic Markers Research Articles

Human epicardial adipose tissue secretome alters metabolomic profile and induces endothelial-to-mesenchymal transition in human lymphatic endothelial cells

Abstract Background Cardiac lymphatic systems play an important role in maintaining myocardial fluid homeostasis during inflammation. We have recently reported the epicardial adipose tissue (EAT)-derived factor induces inflammation and fibrosis in atrial myocardium, which lead to atrial fibrillation (AF). However, the direct effect of EAT on the cardiac lymphatic endothelial cells (LECs) remains unknown. Objective To elucidate the effect of EAT on cardiac LECs, as one of the potential causes of AF. Methods Human EAT samples were collected from 18 consecutive patients with or without AF patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=9, 72.7±8.6 years) and the AF group (n=9, 73.3±7.4 years). Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation to mature adipocytes, cell culture supernatant was collected, and cultured with human LECs. Cell permeability was measured by using FITC-dextran assay. Tube forming ability was analyzed by seeding LECs on the extracellular matrix gels. Metabolomic profiling of LECs was performed by GC-MS system. Results RT-PCR analysis revealed that cell culture supernatant from mature adipocytes of AF patients significantly increased mRNA expression of mesenchymal marker, TAGLN/SM22a (p<0.01) in human LECs, whereas mRNA expression of lymphatic endothelial markers, LYVE1 (p=0.0552) and PROX1 (p=0.0231) were decreased in human LECs, compared to those from mature adipocytes of SR patients. These data suggest cell culture supernatant from mature adipocytes of AF patients highly induce endothelial-to-mesenchymal transition (EndMT) of LECs. When assessing the endothelial barrier function by using FITC-dextran, cell culture supernatant from mature adipocytes of AF patients significantly decreased cell barrier function (p<0.01) compared to those from mature adipocytes of SR patients. Furthermore, cell culture supernatant from mature adipocytes of AF patients significantly decreased the tube forming ability of LECs (p<0.01) compared to those from mature adipocytes of SR patients. To assess the metabolomic changes in human LECs by EAT-secretome, we conducted the GC/MS analysis. Cell culture supernatant from mature adipocytes of AF patients significantly decreased several amino acids including branched-chain amino acid (BCAA; valine, leucine and isoleucine), compared to those from mature adipocytes of SR patients. Conclusion Our study with human EAT and LECs demonstrated that EAT-secretome from AF patients altered metabolomic profile in LECs, and highly caused EndMT in LECs, leading to impaired barrier function and the tube forming ability. Defects of lymphangiogenesis could be a therapeutic target for atrial cardiomyopathy.

Mice with Lymphatic Dysfunction Develop Pathogenic Lung Tertiary Lymphoid Organs that Model an Autoimmune Emphysema Phenotype of COPD.

We have previously shown that mice with loss of C-type lectin-like type II (CLEC2), which have lymphatic dysfunction due to the role of CLEC2 in platelets for maintaining separation between the venous and lymphatic system, develop lung tertiary lymphoid organ (TLO) formation and lung injury that resembles an emphysema phenotype of chronic obstructive pulmonary disease (COPD). We now sought to investigate whether and how TLOs in these mice may play a pathogenic role in lung injury that is relevant to human disease. We found that inhibiting TLO formation using an anti-CD20 antibody in CLEC2-deficient mice partially blocked the development of emphysema. TLOs in CLEC2-deficient mice were rich in plasma cells and were a source of a broad array of autoantibodies. Chronic cigarette smoke exposure increased the size and number of lung TLOs in CLEC2-deficient mice, and was associated with increased markers of antigen presentation and maturation, leading to increased autoantibody deposition. Using lung tissue from COPD patients, we found an increase in lymphatic markers in patients with an emphysema phenotype and autoreactive TLOs compared to COPD patients without emphysema that lack prominent TLOs. Taken together, these results demonstrate that emphysema in mice with lymphatic dysfunction can be partially rescued by blocking TLO formation, and that these TLOs are source of autoantibodies that are exacerbated by cigarette smoke. Our work suggests that lymphatic dysfunction in mice may recapitulate some aspects an autoimmune emphysema phenotype that is seen in a subset of patients with COPD.

Arterial-Lymphatic-Like Endothelial Cells Appear in Hereditary Hemorrhagic Telangiectasia 2 and Contribute to Vascular Leakage and Arteriovenous Malformations.

Arteriovenous malformations (AVMs) are characteristic of hereditary hemorrhagic telangiectasia. Loss-of-function mutations in the activin receptor-like kinase 1 (Alk1) are linked to hemorrhagic telangiectasia type 2. Endothelial-specific deletion of Alk1, endothelial lineage tracing, transcriptomics of single-cell analysis, and electron microscopy were performed to examine the vascular phenotype and characteristics of ALK1-deficient endothelial cells (ECs) after EC-specific Alk1 deletion. Ischemia assays were used to examine the cell capacity for vascular malformation. Connectivity Map with transcriptomic analysis was applied to identify chemical compounds. Specific methods for arteriovenous malformations, such as micro-computed tomography, with other molecular and cell biological tools were also performed. We performed endothelial-specific deletion of Alk1 in mice and found severe arteriovenous malformations and vascular leakage. The transcriptomics of single-cell analysis revealed a new distinctive cell cluster formed after Alk1 deletion where the cells coexpressed arterial and lymphatic endothelial markers. The analysis projected that these cells potentially originated from arterial ECs after Alk1 deletion. This new population was referred to as arterial-lymphatic-like ECs according to its cellular markers, and its appearance was validated in the pulmonary small arteries after Alk1 deletion. Transplantation of these cells caused vascular malformations. Endothelial lineage tracing confirmed that these new arterial-lymphatic-like ECs were derived from ALK1 depleted ECs, potentially arterial ECs. We discovered that SOX17 (SRY-box transcription factor 17) induction was responsible for the derivation of these arterial-lymphatic-like ECs. We showed that direct binding of MDM2 (mouse double minute 2) was required for Sox17 to execute this activity. Inhibition of MDM2 reduced the arteriovenous malformations in the mouse model. Together, our studies revealed the mechanistic underpinnings of ALK1 signaling in regulating the endothelial phenotype and provided possibilities for new therapeutic strategies in hemorrhagic telangiectasia type 2.

Cancer and lymphatic marker FOXC2 drives wound healing and fibrotic tissue formation.

The FOXC2 transcription factor has been tied to a wide range of disease states, serving as a promising prognostic biomarker associated with aggressive basal-like human breast cancers (increased cancer invasion and metastasis). Dysregulation of FOXC2 expression has also been found to promote defects in lymphatic remodeling and hyperplastic lymphedema-distichiasis (LD). Since chronic lymphedema is a forerunner of several malignancies and cancers have been known to arise from poorly healing chronic wounds (e.g., Marjolin ulcers), we examined the effect of Foxc2 dysfunction on skin wound healing. We used our splinted excisional wounding model that mimics human-like wound healing on wildtype and Foxc2+/- mice (n = 4), which demonstrate incomplete lymphatic vasculature and lymphatic dysfunction. Wound size was measured over the course of 18days. Tissue was explanted from both groups at post-operative day (POD) 14 and 18 and stained with Masson's Trichrome to assess scar formation, Picrosirius Red for dermal integrity, or immunofluorescence to assess lymphatic (LYVE1) cell populations. Wildtype mice completely healed by POD 14, while Foxc2+/-mice did not completely heal until POD18. Scar area of healed Foxc2+/-mice (POD 18) was larger than that of healed wild-type mice (POD 14; p = 0.0294). At POD 14, collagen "bers in the scars of Foxc2+/-mice to be narrower (p = 0.0117) and more highly aligned (p = 0.0110), indicating signi"cantly more "brosis in these mice. Collagen "bers in both groups became longer (p = 0.0116) and wider (p = 0.0020) from POD 14 to 18, indicating a temporal evolution of "brosis. Foxc2+/-mice also had lower numbers of LYVE1+, F4/80+ and CD4+ cells compared to wildtype mice. Individuals over 65 years old are more likely to develop cancer and are highly susceptible to developing chronic wounds. Here, we found that FOXC2, which is tied to cancer metastasis and lymphatic dysregulation, also impairs wound healing and promotes "brotic tissue architecture. With FOXC2 proposed as a potential therapeutic target for cancer metastasis, its downstream systemic effects should be considered against the increased chance of developing nonhealing wounds. Further delineation of the microenvironment, cellular events, and molecular signals during normal and Foxc2-associated abnormal wound healing will improve clinical therapies targeting this important marker.

Podoplanin Expression in Early-Stage Colorectal Cancer-Associated Fibroblasts and Its Utility as a Diagnostic Marker for Colorectal Lesions.

(Background) Cancer-associated fibroblasts (CAFs) are major cancer stromal components. CAFs have diverse functions and cell origins. Podoplanin (PDPN), a lymphatic vessel marker, is also a CAF marker in certain cancers. On daily diagnosis of early colorectal carcinoma (CRC), PDPN upregulation in the stroma is often encountered, suggesting PDPN-positive CAFs have emerged. However, PDPN-positive CAFs in early CRC have not been studied well. (Methods) On immunohistochemistry, PDPN expression in the lamina propria or stroma of adenomas, early CRCs, and neuroendocrine tumors, their normal neighbors, and non-neoplastic colorectal lesions were compared. Single-cell RNA sequencing (scRNA-seq) of CRC was used to explore PDPNhigh CAFs' cell origins. (Results) Reticular cells or pericryptal fibroblasts in the lamina propria of adenomas and early CRCs showed higher PDPN expression than did normal mucosae and non-neoplastic lesions (p < 0.01). Pericryptal PDPN expression was a diagnostic feature of adenomas and early CRCs. scRNA-seq of CRCs highlighted that PDPNhigh CAFs had distinctly higher COL4A1, COL4A2, and WNT5A expression, unlike well-known CAFs characterized by high FAP, POSTN, or ACTA2 expression. (Conclusions) We demonstrated that pericryptal fibroblasts and reticular cells in the lamina propria are origins of early-stage CRC CAFs and thus have potential as a diagnostic marker for distinguishing colorectal non-neoplastic from neoplastic lesions.

Multi-parameter tunable synthetic matrix for engineering lymphatic vessels

Controlling the formation of 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, little is known about the molecular regulation that controls lymphatic tube formation in a synthetic matrix. In this study, we utilize hyaluronic acid (HA)-hydrogels to design a novel platform for decoupled investigation into how mechanical and biochemical cues regulate lymphatic vessel formation in a synthetic matrix. Using HA and controlling the degree of modification provides a method to preserve and modulate key lymphatic markers Prox1, LYVE-1, and Pdpn. The chemistry of the system allows for spatial and temporal patterning of specific peptides and substrate stiffnesses, and an MMP-sensitive crosslinker allowed cells to degrade and remodel their matrix. Through systematic optimization of multiple parameters, we have designed a system that allows human lymphatic endothelial cells (LECs) to self-assemble into vessels in vitro within 3 days. These engineered vessels can be cultured for up to 3 weeks and can be used for high-throughput mechanistic studies, or can be implanted into immunodeficient mice where they have demonstrated the ability to integrate and mature. Collectively, these studies report a novel, fully-defined 3D synthetic matrix system capable of generating lymphatic vessels in vitro that provide promise as an in vitro screening platform and as a therapeutic vessel transplant, which to our knowledge, is the first ever 3D lymphatic tissue engineering approach to not require the use of support cells.

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.

Acute kidney injury results in long-term alterations of kidney lymphatics in mice.

The long-term effects of a single episode of acute kidney injury (AKI) induced by bilateral ischemia-reperfusion injury (BIRI) on kidney lymphatic dynamics are not known. The purpose of this study was to determine if alterations in kidney lymphatics are sustained in the long term and how they relate to inflammation and injury. Mice underwent BIRI as a model of AKI and were followed up to 9 mo. Although kidney function markers normalized following initial injury, histological analysis revealed sustained tissue damage and inflammation for up to 9 mo. Transcriptional analysis showed both acute and late-stage lymphangiogenesis, supported by increased expression of lymphatic markers, with unique signatures at each phase. Expression of Ccl21a was distinctly upregulated during late-stage lymphangiogenesis. Three-dimensional tissue cytometry confirmed increased lymphatic vessel abundance, particularly in the renal cortex, at early and late timepoints postinjury. In addition, the study identified the formation of tertiary lymphoid structures composed of CCR7+ lymphocytes and observed changes in immune cell composition over time, suggesting a complex and dynamic response to AKI involving tissue remodeling and immune cell involvement. This study provides new insights into the role of lymphatics in the progression of AKI to chronic kidney disease.NEW & NOTEWORTHY Here, we perform the first, comprehensive study of long-term lymphatic dynamics following a single acute kidney injury (AKI) event. Using improved three-dimensional image analysis and an expanded panel of transcriptional markers, we identify multiple stages of lymphatic responses with distinct transcriptional signatures, associations with the immune microenvironment, and collagen deposition. This research advances kidney lymphatic biology, emphasizing the significance of longitudinal studies in understanding AKI and the transition to chronic kidney disease.

Abstract P346: Expansion of meningeal lymphatic vessels in DOCA-salt hypertension

The meningeal lymphatic system is critical for proper brain waste clearance and maintenance of the local meningeal neuroimmune niche. Meningeal lymphatic function is impaired during aging and neurodegenerative diseases and is associated with the development of cognitive impairment. There is a bidirectional communication between meningeal lymphatic function and dural T cells, whereby the drainage of T cells into the deep cervical lymph nodes depends on functional lymphatic vessels, and meningeal lymphatic function is itself modulated by T cell derived cytokines. We have previously shown that IL-17A producing T cells accumulate in the dura during deoxycorticosterone acetate (DOCA)-salt hypertension and contribute to the impairment of neurovascular coupling and cognitive function. However, the mechanisms by which these T cells accumulate in the dura remain to be determined. Here, we tested the hypothesis that the meningeal lymphatic vessels are impaired during DOCA-salt hypertension, thus contributing to dural T cell accumulation. 12-week old C57BL6 male mice were implanted with a s.c. DOCA pellet and received 0.9% NaCl in the drinking water for 21 days (n=4). Control mice underwent a sham surgery (n=4). Blood pressure was monitored by tail-cuff plethysmography. Meningeal lymphatic vessels were assessed by immunohistochemistry using the lymphatic vessel marker LYVE1. In a separate group of mice, we assessed meningeal lymphatic function by delivering Alexa647-Ovalbumin or IgG-RPE into the cisterna magna and measuring fluorescence in the deep cervical lymph nodes one hour after injection. DOCA-salt mice developed increased systolic blood pressure (control: 117.7 ± 4.9 vs DOCA: 141.5 ± 2.9mmHg). We observed no difference in the CD31+ dural sinus area between control and DOCA-salt (control: 11.482 ± 0.718 vs DOCA: 11.816 ± 0.662 mm 2 ). We found a significant increase in LYVE1+ area surrounding the dural sinuses in DOCA-salt (control: 0.508 ± 0.039 vs DOCA: 0.816 ± 0.105 mm 2 ; p=0.0329 by unpaired two-tailed t-test) and an increase in LYVE1+/CD31+ % area (control: 4.476 ± 0.462 vs DOCA: 6.840 ± 0.609 %; p=0.0213 by unpaired two-tailed t-test) which was not different between the sagittal sinus or the transverse sinus. Contrary to our hypothesis, our data suggests that the meningeal lymphatic vessels are expanded, not retracted, during DOCA-salt hypertension. The interaction between meningeal lymphatics and dura immunity in hypertension remains to be determined.

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.

Brain lymphatic vessels accompanying meningeal arteries in the human

IntroductionRecent studies showed the connection between cerebrospinal fluid (CSF) and deep cervical lymph nodes (dCLN) via lymphatic vessels (LV)s. These LVs accompany dural sinuses, cranial nerves, and arteries. The present study aimed to show in addition to the main trunk of the middle meningeal artery (MMA) whether the anterior and posterior branches of MMA; the anterior (AMA) and posterior (PMA) meningeal arteries also have a role in brain lymphatic drainage in humans. Materials and methodSamples of the middle meningeal artery (MMA) and its two main branches (anterior and posterior), anterior and posterior meningeal arteries (AMA and PMA) were obtained from 3 cadavers and 6 autopsies. Podoplanin (PDPN) (lymphatic endothelial marker) and CD31 (vascular endothelial marker) immunohistochemistry (IHC) were applied to 5 μm thick transverse sections and counterstained with hematoxylin. Furthermore, western blotting techniques were applied to the same tissue samples in 3 autopsy cases to demonstrate the presence of PDPN. ResultsWe observed the presence of the LVs along the main trunk of MMA and its two branches, AMA, and PMA. The LVs along the MMA (main trunk) had a larger diameter range than its branches and the AMA and PMA. The diameter of LVs of the PMA was larger than AMA. Furthermore, fluid channels with varying diameters and densities were closely localized to the LVs. The western blot PDPN protein expression confirmed IHC results. ConclusionThe LVs along the main trunk, anterior and posterior branches of the MMA, AMA, and PMA also provide an extracranial pathway for thought drainage of waste from the brain tissue in humans. Thus, the organization of the extracranial transport of LVs of the brain should be well-defined for understanding the role of lymphatic drainage in various neurological diseases.

#1536 Serum soluble LYVE1 is a promising non-invasive biomarker of renal fibrosis: a population-based retrospective cross-sectional study

Abstract Background and Aims Diagnosis of renal fibrosis can only be verified by kidney biopsy, but biomarkers for non-invasive evaluation remain unsatisfactory. Patients with fibrosis often have abnormalities of the lymphatic vascular system and associated immune function. We describe here a lymphatic marker as a candidate biomarker for fibrosis. Method After assessing and grading the fibrosis scores, testing serum soluble lymphatic vessel endothelial hyaluronan receptor1 (sLYVE1) level, and collecting clinical information, the association between sLYVE1 and renal fibrosis was analyzed. Logistic regression analysis was used to screen variables. Diagnosis models with or without sLYVE1 were built, and nomograms were plotted. Calibration curve, C-index, and DCA were performed to assess the models. Results A total of 298 patients were enrolled in the study, of which 199 were included in the training cohort and 99 patients in the validation cohort. Serum sLYVE1 levels markedly elevated with increasing fibrosis grade (p &amp;lt; 0.05). ROC analysis of sLYVE1 showed an AUC of 0.791 and 0.846 with optimal cut-off value of 405.25 ng/mL and 498.55 ng/mL for the prediction of moderate-to-severe renal fibrosis (MSF) and severe renal fibrosis (SF), respectively (Fig. 1). The diagnostic nomogram model without sLYVE1 (model 1) included traditional clinical determinants (C-index: 0.658 for MSF; 0.603 for SF). A combination of model 1 and sLYVE1 (model 2) improved predictive performance (C-index: 0.847 for MSF; 0.856 for SF) (Figs 2 and 3). Calibration curve and DCA demonstrated a better consistency accuracy and clinical benefit of model 2 than model 1. Conclusion Serum sLYVE1 may be identified as a potential biomarker of renal fibrosis. Models incorporating sLYVE1 may be beneficial for a more accurate non-invasive diagnosis of renal fibrosis.

VEGF-C and 5-Fluorouracil Improve Bleb Survival in a Rabbit Glaucoma Surgery Trabeculectomy Model.

To evaluate VEGF-C-induced lymphoproliferation in conjunction with 5-fluorouracil (5-FU) antimetabolite treatment in a rabbit glaucoma filtration surgery (GFS) model. Thirty-two rabbits underwent GFS and were assigned to four groups (n = 8 each) defined by subconjunctival drug treatment: (a) VEGF-C combined with 5-FU, (b) 5-FU, (c) VEGF-C, (d) and control. Bleb survival, bleb measurements, and IOP were evaluated over 30days. At the end, histology and anterior segment OCT were performed on some eyes. mRNA was isolated from the remaining eyes for RT-PCR evaluation of vessel-specific markers (lymphatics, podoplanin and LYVE-1; and blood vessels, CD31). Qualitatively and quantitatively, VEGF-C combined with 5-FU resulted in blebs which were posteriorly longer and wider than the other conditions: vs. 5-FU (P = 0.043 for longer, P = 0.046 for wider), vs. VEGF-C (P<0.001, P < 0.001) and vs. control (P<0.001, P<0.001). After 30days, the VEGF-C combined with 5-FU condition resulted in longer bleb survival compared with 5-FU (P = 0.025), VEGF-C (P<0.001), and control (P<0.001). Only the VEGF-C combined with 5-FU condition showed a negative correlation between IOP and time that was statistically significant (r = -0.533; P = 0.034). Anterior segment OCT and histology demonstrated larger blebs for the VEGF-C combined with 5-FU condition. Only conditions including VEGF-C led to increased expression of lymphatic markers (LYVE-1, P<0.001-0.008 and podoplanin, P = 0.002-0.011). Expression of CD31 was not different between the groups (P = 0.978). Adding VEGF-C lymphoproliferation to standard antimetabolite treatment improved rabbit GFS success and may suggest a future strategy to improve human GFSs.

Stromal Vascular Fraction Derived Vasculogenesis Is Associated with the Formation of Malformed Lymphatic Vessel Structure

Tissue engineering and the development of therapies aimed at manipulating microvascular function require the ability to generate both blood and lymphatic vessels. Adipose-derived stromal vascular fraction (SVF), consisting of endothelial cells, stem cells, pericytes, smooth muscle cells, fibroblasts and immune cells, has emerged as a potential heterogeneous stem cell population for de novo vessel growth. SVF derived vasculogenesis has been reported to result in the dramatic formation of blood vessel segments that can connect with host vasculatures. Understanding how SVF can be used to therapeutically form vessels requires 1) exploration of the cell dynamics involved in the vasculogenesis process and 2) cauterization of the vessel structures. The objective of this study was to characterize SVF de novo vessel formation in an intact adult murine tissue environments. SVF was isolated from subcutaneous adipose in the inguinal region from adult C57BL/6 mice, labeled with DiI and seeded onto avascular mesentery tissues and then cultured in MEM + 10% fetal bovine serum (FBS) for up to 5 days (35 μl SVF cells/tissue; isolated from 1.8 g inguinal fats and resuspended in 200 μl MEM+FBS). Tissues were then labeled for PECAM (endothelial marker) + Lyve-1 (lymphatic marker) or NG2 (pericyte marker). By day 1, individual SVF cells started to form multi-cellular PECAM positive chord structures. Day 3 tissues displayed apparent vessel structures characterized by asterisk shaped clusters displaying a central PECAM positive “hub” and radial “spoke-like” extensions. At later time points and in regions of increased vascular coverage, the vessel structures were connected in a network pattern. In additional studies for which angiogenesis was pre-stimulated in the normally avascular mouse mesenteries, the SVF derived vessels connected to the host networks. A subset of structures during the vasculogenesis process displayed blunt ended lymphatic-like vessel structures termed “blebs.” The endothelial cell “blebs” were characterized by large, irregular diameters and a mosaic of Lyve-1 positive and negative cells. The “blebs” were also observed to connect with nearby SVF derived PECAM positive vessel segments. Our evidence of PECAM positive hub and lymphatic-like blebs provoke questions about SVF derived vessel function and suggest a previously undiscovered potential for SVF to form lymphatic vessel structures. NIH grant R21HL159501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1064: Creation Of Lymphatic Endothelial Cells From Induced Pluripotent Stem Cells For Use In Regenerative Therapy For Lymphedema

Background/Objective: Lymphedema is a condition characterized by impaired flow of interstitial fluid through the lymphatic system and chronic obliteration of distal lymphatic collecting vessels. Despite advances in our knowledge of the development of lymphatic vessels, there has been little progress translating this knowledge into clinically relevant regenerative therapies. Although lymphatic endothelial cells (LECs) share a lineage with arterial and venous endothelial cell progenitors, currently described processes to derive LECs from stem cells in vitro take up to a month, require embryoid body formation, and have been minimally characterized. Methods: We have developed a protocol for the generation of lymphatic endothelial cells from induced pluripotent stem cells (iPSCs) over 11 days. First, iPSCs are directed to become endothelial cell progenitors over 6 days and purified with CD144 magnetic beads. Then, ECs are directed to differentiate into LECs using a 5-day treatment with 10 ng/mL Ang1 alone or in combination with 200 ng/mL VEGFc. Human primary LECs were used as a positive control and iPSC-ECs without Ang1 or VEGFc treatment as well as fibroblasts were negative controls. Results: Treating iPSC-derived endothelial cells for 5 days with Ang1 +/- VEGFc yields iPSC-LECs with morphological similarity to primary lymphatic controls and expression of multiple lymphatic markers including Podoplanin, Lyve1, Prox1, and VEGFR3, verified by immunofluorescent staining, western blotting, and flow cytometry across two independent cell lines. In flow cytometry studies, 75% of LECs treated with Ang1 and 72% of LECs treated with Ang1 and VEGFc co-express Podoplanin and Lyve1. There was more than a three-fold increase compared to CD144+ endothelial progenitors (20.4%, p&lt;0.001) while fibroblast expression was negligible (0.6%, p&lt;0.001). Finally, the overall transcriptome of LECs treated with Ang1 was compared to primary human lymphatic cells using single cell RNA sequencing. Conclusions: Treatment with Ang1 with or without VEGFc allows fast and efficient generation of LECs from iPSCs. This allows LEC differentiation in vitro from a wide variety of patient specific cell types and improves the practicality of regenerative therapy for lymphedema.

MRC1 and LYVE1 expressing macrophages in vascular beds of GNAQ p.R183Q driven capillary malformations in Sturge Weber syndrome

Sturge-Weber syndrome (SWS), a neurocutaneous disorder, is characterized by capillary malformations (CM) in the skin, brain, and eyes. Patients may suffer from seizures, strokes, and glaucoma, and only symptomatic treatment is available. CM are comprised of enlarged vessels with endothelial cells (ECs) and disorganized mural cells. Our recent finding indicated that the R183Q mutation in ECs leads to heightened signaling through phospholipase Cβ3 and protein kinase C, leading to increased angiopoietin-2 (ANGPT2). Furthermore, knockdown of ANGPT2, a crucial mediator of pro-angiogenic signaling, inflammation, and vascular remodeling, in EC-R183Q rescued the enlarged vessel phenotype in vivo. This prompted us to look closer at the microenvironment in CM-affected vascular beds. We analyzed multiple brain histological sections from patients with GNAQ-R183Q CM and found enlarged vessels devoid of mural cells along with increased macrophage-like cells co-expressing MRC1 (CD206, a mannose receptor), CD163 (a scavenger receptor and marker of the monocyte/macrophage lineage), CD68 (a pan macrophage marker), and LYVE1 (a lymphatic marker expressed by some macrophages). These macrophages were not found in non-SWS control brain sections. To investigate the mechanism of increased macrophages in the perivascular environment, we examined THP1 (monocytic/macrophage cell line) cell adhesion to EC-R183Q versus EC-WT under static and laminar flow conditions. First, we observed increased THP1 cell adhesion to EC-R183Q compared to EC-WT under static conditions. Next, using live cell imaging, we found THP1 cell adhesion to EC-R183Q was dramatically increased under laminar flow conditions and could be inhibited by anti-ICAM1. ICAM1, an endothelial cell adhesion molecule required for leukocyte adhesion, was strongly expressed in the endothelium in SWS brain histological sections, suggesting a mechanism for recruitment of macrophages. In conclusion, our findings demonstrate that macrophages are an important component of the perivascular environment in CM suggesting they may contribute to the CM formation and SWS disease progression.

The development of early human lymphatic vessels as characterized by lymphatic endothelial markers

Lymphatic vessel development studies in mice and zebrafish models have demonstrated that lymphatic endothelial cells (LECs) predominantly differentiate from venous endothelial cells via the expression of the transcription factor Prox1. However, LECs can also be generated from undifferentiated mesoderm, suggesting potential diversity in their precursor cell origins depending on the organ or anatomical location. Despite these advances, recapitulating human lymphatic malformations in animal models has been difficult, and considering lymphatic vasculature function varies widely between species, analysis of development directly in humans is needed. Here, we examined early lymphatic development in humans by analyzing the histology of 31 embryos and three 9-week-old fetuses. We found that human embryonic cardinal veins, which converged to form initial lymph sacs, produce Prox1-expressing LECs. Furthermore, we describe the lymphatic vessel development in various organs and observe organ-specific differences. These characterizations of the early development of human lymphatic vessels should help to better understand the evolution and phylogenetic relationships of lymphatic systems, and their roles in human disease.

Vitamin D accelerates the subdural hematoma clearance through improving the meningeal lymphatic vessel function.

Subdural hematoma (SDH) drains into the extracranial lymphatic system through the meningeal lymphatic vessels (mLVs) but the formation of SDH impairs mLVs. Because vitamin D (Vit D) can protect the endothelial cells, we hypothesized that Vit D may enhance the SDH clearance. SDH was induced in Sprague-Dawley rats and treated with Vit D or vehicle. Hematoma volume in each group was measured by H&E staining and hemoglobin quantification. Evans blue (EB) quantification and red blood cells injection were used to evaluated the drainage of mLVs. Western blot analysis and immunofluorescence were conducted to assess the expression of lymphatic protein markers. We also examined the inflammatory factors levels in subdural space by ELISA. Vit D treatment significantly reduced SDH volume and improved the drainage of SDH to cervical lymph nodes. The structure of mLVs in SDH rats were protected by Vit D, and the expressions of LYVE1, PROX1, FOXC2, and VE-cadherin were increased after Vit D treatment. The TNF-α, IL-6, and IL-8 levels were reduced in Vit D group. In vitro, Vit D also increased the VE-cadherin expression levels under inflammation. Vit D protects the structure of mLVs and enhances the absorption of SDH, partly by the anti-inflammatory effect of Vit D.

Novel erectile analyses revealed augmentable penile Lyve-1, the lymphatic marker, expression.

The pathophysiology of penis extends to erectile dysfunction (ED) to conditions including sexually transmitted diseases (STDs) and cancer. To date, there has been little research evaluating vascular drainage from the penis. We aimed to evaluate penile blood flow invivo and analyze its possible relationship with the lymphatic maker. We established an invivo system designed to assess the dynamic blood outflow from the corpus cavernosum (CC) by dye injection. To analyze lymphatic characteristics in the CC, the expression of Lyve-1, the key lymphatic endothelium marker, was examined by the invitro system and lipopolysaccharide (LPS) injection to mimic the inflammatory conditions. A novel cavernography methods enable high-resolution morphological and functional blood drainage analysis. The expression of Lyve-1 was detected along the sinusoids. Furthermore, its prominent expression was also observed after penile LPS injection and in the erectile condition. The current invivo system will potentially contribute to the assessment of penile pathology from a novel viewpoint. In addition, current analyses revealed inducible Lyve-1 expression for LPS injection and the erection state, which requires further analyses on penile lymphatic system.

Comparative Analysis of Vascular Cell Differentiation From Peripheral Blood Mononuclear Cell‐ and Urine‐Derived Induced Pluripotent Stem Cells

Background and Objectives: Peripheral blood mononuclear cells (PBMCs) and urine‐derived epithelial cells have both emerged as valuable sources for induced pluripotent stem cell (iPSC) generation, each presenting unique advantages in terms of accessibility and reprograming efficiency. This study aimed to assess and compare the potential of PBMC‐derived iPSCs (PiPSCs) and urine‐derived iPSCs (UiPSCs) in generating functional endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), which are critical for vascular tissue engineering and disease modeling. Phenotypic characteristics, differentiation efficacy, and functional properties of iPSC‐derived ECs and VSMCs from these distinct sources were investigated to reveal variations attributed to cellular origin.Methods and Results: PiPSCs and UiPSCs both successfully differentiated into functional ECs and VSMCs. EC differentiation efficiency was similar, yielding about 45% mature ECs with characteristic morphology, marker expression, and tube formation abilities, showing no significant differences between cell types. Transcriptomic analysis revealed upregulation of key endothelial markers (platelet endothelial cell adhesion molecule 1 [PECAM1], cadherin 5 [CDH5], and melanoma cell adhesion molecule [MCAM]) and downregulation of lymphatic markers (Fms‐related tyrosine kinase 4 [FLT4], prospero homeobox protein 1 [PROX1], and podoplanin [PDPN]), confirming blood EC identity. The upregulation of collagen type IV alpha 1 chain (COL4A1) and collagen type I alpha 1 chain (COL1A1) indicated a mature endothelial state with enhanced extracellular matrix (ECM) production. VSMC differentiation resulted in high percentages of α‐smooth muscle actin (α‐SMA) positive cells for both PiPSCs (96%) and UiPSCs (94%). These VSMCs exhibited typical spindle‐shaped morphology, expressed VSMC markers, and responded to carbachol. Transcriptomic analysis showed significant upregulation of VSMC markers (actin alpha 2 [ACTA2], caldesmon 1 [CALD1], calponin 1 [CNN1], transgelin [TAGLN], tropomyosin 2 [TPM2]), with concurrent downregulation of ACTA1 and upregulation of ACTA2, confirming their vascular smooth muscle phenotype. The upregulation of COL6A1 in VSMCs indicated a mature phenotype with enhanced ECM production, crucial for vascular tissue integrity and function. Gene set enrichment analysis highlighted the upregulation of multiple hallmark pathways, delineating a distinctive transcriptional profile.Conclusions: This study presents a comprehensive comparative analysis of functionally differentiated ECs and VSMCs derived from PiPSCs and UiPSCs, providing critical insights into the expression patterns and phenotypic transitions during differentiation. Our findings enhance the understanding of distinct molecular signatures in iPSCs from different sources and their progeny.