Lymphatic Vessel Endothelial Hyaluronan Receptor Research Articles

VEGFC Overexpression in Kidney Progenitor Cells Is a Model of Renal Lymphangiectasia.

Lymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease. As injury-related lymphangiogenesis is driven by signaling from the receptor VEGFR3 (vascular endothelial growth factor receptor 3) in response to the cognate growth factor VEGF (vascular endothelial growth factor)-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remain poorly defined. We generated a new mouse model expressing Vegfc under regulation of the nephron progenitor Six2Cre driver strain (Six2Vegfc). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and 3-dimensional imaging. Six2Vegfc mice had reduced body weight and kidney function compared with littermate controls. Six2Vegfc kidneys demonstrated large peripelvic fluid-filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. Three-dimensional imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+ (lymphatic vessel endothelial hyaluronan receptor-1)/PDPN+ (podoplanin)/VEGFR3+ lymphatic capillaries extending alongside EMCN+ (endomucin) peritubular capillaries. There was no change in EMCN+ peritubular capillary density. Kidney lymphatic density was robustly increased in the Six2Vegfc mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR3. The model resulted in malformation of the lymphatic hilar plexus, resulting in severe hydronephrosis that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGFC signaling during kidney development and provides new insight into human renal lymphatic malformations.

Distinct activation of M1 and M2 macrophages in the primary pterygium lymphangiogenesis

The precise role and innate immunological mechanisms underlying lymphangiogenesis in pterygium remain unclear. This study aimed to investigate the presence of M1 and M2 macrophages and their correlation with pro-lymphangiogenic activation and lymphatic endothelial expression in human pterygium stromal tissues. We analyzed human pterygium and subject-matched normal conjunctival tissues for the expression of these factors and conducted in vitro experiments to assess interactions between macrophages and pterygium fibroblasts. Myeloid and M1 macrophage markers were upregulated in pterygium. M1 macrophages were associated with the upregulation of pro-lymphangiogenic vascular endothelial growth factor C (Vegfc) in pterygium tissues and induced inflammatory signals in pterygium fibroblasts. In contrast, lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) expression was associated with M2 markers but not with M1 markers. Notably, the clinical severity of pterygium was inversely correlated with the expression of the M2 marker Cd163. These findings suggest that M1 and M2 macrophages play distinct roles in the pathogenesis of pterygium, with M1 macrophages enhancing lymphangiogenic stimulation and inflammatory responses, while M2 macrophages are associated with Lyve1 expression and reduced severity of pterygium. Understanding these mechanisms may advance our current understanding of lymphatic biology in pterygium.

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.

Urine biomarkers can predict prostate cancer and PI-RADS score prior to biopsy

Prostate-Specific Antigen (PSA) based screening of prostate cancer (PCa) needs refinement. The aim of this study was the identification of urinary biomarkers to predict the Prostate Imaging—Reporting and Data System (PI-RADS) score and the presence of PCa prior to prostate biopsy. Urine samples from patients with elevated PSA were collected prior to prostate biopsy (cohort = 99). The re-analysis of mass spectrometry data from 45 samples was performed to identify urinary biomarkers to predict the PI-RADS score and the presence of PCa. The most promising candidates, i.e. SPARC-like protein 1 (SPARCL1), Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), Alpha-1-microglobulin/bikunin precursor (AMBP), keratin 13 (KRT13), cluster of differentiation 99 (CD99) and hornerin (HRNR), were quantified by ELISA and validated in an independent cohort of 54 samples. Various biomarker combinations showed the ability to predict the PI-RADS score (AUC = 0.79). In combination with the PI-RADS score, the biomarkers improve the detection of prostate carcinoma-free men (AUC = 0.89) and of those with clinically significant PCa (AUC = 0.93). We have uncovered the potential of urinary biomarkers for a test that allows a more stringent prioritization of mpMRI use and improves the decision criteria for prostate biopsy, minimizing patient burden by decreasing the number of unnecessary prostate biopsies.

#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 < 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.

Serum proteomic profiling of patients with compensated advanced chronic liver disease with and without clinically significant portal hypertension.

Portal hypertension (PH) drives the progression of liver cirrhosis to decompensation and death. Hepatic venous pressure gradient (HVPG) measurement is the standard of PH quantification, and HVPG≥10 mmHg defines clinically significant PH (CSPH). We performed proteomics-based serum profiling to search for a proteomic signature of CSPH in patients with compensated advanced chronic liver disease (cACLD). Consecutive patients with histologically confirmed cACLD and results of HVPG measurements were prospectively included. Serum samples were pooled according to the presence/absence of CSPH and analysed by liquid chromatography-mass spectrometry. Gene set enrichment analysis was performed, followed by comprehensive literature review for proteins identified with the most striking difference between the groups. We included 48 patients (30 with, and 18 without CSPH). Protein CD44, involved in the inflammatory response, vascular endothelial growth factor C (VEGF-C) and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), both involved in lymphangiogenesis were found solely in the CSPH group. Although identified in both groups, proteins involved in neutrophil extracellular traps (NET) formation, as well as tenascin C, autotaxin and nephronectin which mediate vascular contractility and lymphangiogenesis were more abundant in CSPH. We propose that altered inflammatory response, including NET formation, vascular contractility and formation of new lymph vessels are key steps in PH development. Proteins such as CD44, VEGF-C, LYVE-1, tenascin C, Plasminogen activator inhibitor 1, Nephronectin, Bactericidal permeability-increasing protein, Autotaxin, Myeloperoxidase and a disintegrin and metalloproteinase with thrombospondin motifs-like protein 4 might be considered for further validation as potential therapeutic targets and candidate biomarkers of CSPH in cACLD.

Abstract 159: LYVE-1-expressing macrophages modulate the hyaluronan-containing extracellular matrix in the mammary stroma and contribute to mammary tumor growth

Abstract Macrophages represent a heterogeneous myeloid population with diverse functions in normal tissues and tumors. While macrophages expressing the cell surface marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) have been identified in stromal regions of the normal mammary gland and in the peri-tumoral stroma, their functions within these regions are not well-understood. Using a genetic LYVE-1+ macrophage deletion mouse model, we demonstrate that loss of LYVE-1+ macrophages is associated with altered extracellular matrix remodeling in the normal mammary gland and reduced mammary tumor growth in vivo. In further studies focused on investigating the functions of LYVE-1+ macrophages in the tumor microenvironment, we demonstrate that LYVE-1 expression correlates with an increased ability of macrophages to bind, internalize, and degrade hyaluronan. Consistent with this, we show that deletion of LYVE-1+ macrophages correlates with increased hyaluronan accumulation in both the normal mammary gland and in mammary tumors. Analysis of single cell RNA sequencing of macrophages isolated from these tumors reveals that deletion of LYVE-1+ macrophages in tumors drives a shift in the majority of the remaining macrophages towards a pro-inflammatory phenotype, as well as an increase in CD8+ T cell infiltration. Together, these findings demonstrate that LYVE-1+ macrophages represent a tumor-promoting anti-inflammatory subset of macrophages that contributes to hyaluronan remodeling in the tumor microenvironment. Citation Format: Alexis K. Elfstrum, Annisa H. Rumahorbo, Braedan M. McCluskey, Kathryn L. Schwertfeger. LYVE-1-expressing macrophages modulate the hyaluronan-containing extracellular matrix in the mammary stroma and contribute to mammary tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 159.

Networks of Nerve Fibers, and Blood and Lymphatic Vessels in the Mouse Auricle: The Structural Basis of Ear Acupuncture.

Ear acupuncture, as a system for treating and preventing diseases through stimulation of points on the auricle, has been systematically introduced during the last 60 years. Although the auricular cartography was described somatotopically as an inverted fetus by Paul Nogier, MD, the underlying mechanism of auricular stimulation remains unclear. The aim of this research was to gain an understanding of the structural basis of auricular stimulation, as well as showing the distribution of the nerve fibers, and the blood and lymphatic vessels. The distribution of nerve fibers, and blood and lymphatic vessels was examined in whole-mount auricular skins of mice by combining the biomarkers protein gene product 9.5, cluster of differentiation 31, and lymphatic-vessel endothelial hyaluronan receptor-1 following tissue-clearing treatment with multiple immunofluorescent staining. The labeled nerve fibers, and the blood and lymphatic vessels were distributed extensively in the inner and outer parts of the auricular skin. Auricular nerves aligning with blood vessels ran from the basal region to the peripheral region and crossed over lymphatic vessels, thus forming the neural, vascular, and lymphatic networks. As these are important tissue components of auricular skin, this result implies that the auricular nerve fibers, and blood and lymphatic vessels may coordinate with each other to respond directly to auricular stimulation.

Role of lymphatic endothelium specific hyaluronan receptor 1 in virus infection and associated diseases.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) serves as a prominent marker for lymphatic endothelial cells (LECs) and is pivotal in the process of lymphangiogenesis, a critical factor in cancer development and metastasis. Overexpression of LYVE-1 has been observed in various cancers, where it is recognized as an adverse prognostic indicator. Targeting LYVE-1 has demonstrated inhibitory effects on tumor cell proliferation, migration, and the formation of lymph node metastases both in vitro and in vivo. While extensive research has focused on the role of LYVE-1 in cancer cells, its involvement in virus infection and associated diseases remains largely unexplored. This review consolidates recent findings regarding the expression of LYVE-1 and its functions in lymphangiogenesis during various viral infections and the development of related diseases, with a particular emphasis on Kaposi's sarcoma herpesvirus. Despite the limited available data, it is evident that further studies are essential to comprehensively understand the contribution of LYVE-1 to viral pathogenesis and oncogenesis.

Histological and biochemical changes in lymphatic vessels after skeletal muscle injury induced by lengthening contraction in male mice.

Lymphatic vessels are actively involved in the recovery process of inflamed tissues. However, the changes in intramuscular lymphatic vessels during inflammation caused by skeletal muscle injury remain unclear. Therefore, the purpose of this study was to clarify the changes in lymphatic vessels after skeletal muscle injury. The left tibialis anterior muscles of male mice were subjected to lengthening contractions (LC) for inducing skeletal muscle injury, and samples were collected on Days 2, 4, and 7 for examining changes in both the skeletal muscles and intramuscular lymphatic vessels. With hematoxylin-eosin staining, the inflammatory response was observed in myofibers on Days 2 and 4 after LC, whereas regeneration of myofibers was found on Day 7 after LC. The number and area of intramuscular lymphatic vessels analyzed by immunohistochemical staining with an antibody against lymphatic vessel endothelial hyaluronan receptor 1 were significantly increased only on Day 4 after LC. Based on the abovementioned results, intramuscular lymphatic vessels undergo morphological changes such as increase under the state of muscle inflammation. This study demonstrated that the morphology of intramuscular lymphatic vessels undergoes significant changes during the initial recovery phase following skeletal muscle injury.

Immunologic landscape of human hepatic hemangiomas and epithelioid hemangioendotheliomas.

The missing requirement for resection for the majority of hepatic hemangiomas (HH) and tissue scarcity for rare diseases such as hepatic epithelioid hemangioendotheliomas (HEHE) complicate the characterization of the spatial immunovascular niche of these benign and malignant vascular neoplastic diseases. Two tissue cohorts containing 98 HHs and 13 HEHEs were used to study entity-specific and disease stage-specific endothelial cell (EC) phenotype and immune cell abundance. Using semiquantitative assessment, annotation-based cell classifiers, digital cell detection on whole slides, and tissue microarrays, we quantified 23 immunologic and vascular niche-associated markers and correlated this with clinicopathologic data. Both HH and HEHE ECs were characterized by a CD31high, CD34high, FVIII-related antigenhigh expression phenotype with entity-specific expression differences of sinusoidal EC markers Stabilin1, Stabilin2, CD32, and Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1). Cell detection identified an HH margin-prevailing immunologic response dominated by Myeloperoxidase+ (MPO+) macrophages, CD3+ and CD8+ T cell subsets, and B cells (CD20+, CD79A+). In HEHE, increased CD68+ and CD20+ cell demarcation of lesion margins was observed, while CD3+ and CD8+ T cells were equally detectable both marginally and intralesionally. Stage-specific pairwise correlation analysis of HH and HEHE revealed disease entity-specific immunologic infiltration patterns as seen by high CD117+ cell numbers in HH, while HEHE samples showed increased CD3+ T cell infiltration. ECs in HH and HEHE share a continuous EC expression phenotype, while the expression of sinusoidal EC markers is more highly retained in HEHE. These phenotypic differences are associated with a unique and disease-specific immunovascular landscape.

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

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. 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. 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<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. 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). Calibration curve and DCA demonstrated a better consistency accuracy and clinical benefit of model 2 than model 1. 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.

Abstract A013: Targeting perivascular macrophages with an orally bioavailable heme oxygenase-1 inhibitor improves responses to chemotherapeutic drugs in cancer

Abstract The tumor microenvironment (TME) plays a crucial role in cancer progression. It consists of various elements including stromal cells, proteins, and physical factors that support the growth and spread of cancer. Targeting the TME has become an appealing strategy for cancer treatment. Tumor-associated macrophages (TAMs) are an abundant stromal cell type in the TME and, due to their plasticity, TAMs can be exploited by cancer cells and the broader stromal reaction to promote a pro-tumoral response. TAMs have been implicated in facilitating a variety of pro-tumoral pathways including resistance to anti-cancer therapeutics, immune suppression, angiogenesis, and metastasis. Our group has recently characterized a subpopulation of pro-angiogenic and immune-suppressive TAMs which can be identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). LYVE-1+ TAMs reside spatially proximal to blood vasculature and adopt a collaborative multi-cellular ‘nest’ arrangement which may support their biological activity. LYVE-1+ TAMs selectively express high levels of the immunomodulatory enzyme heme oxygenase-1 (HO-1), an enzyme which catabolizes heme to generate the biologically active catabolites carbon monoxide (CO), biliverdin and ferrous iron. Genetic inactivation of HO-1 in LYVE-1+ TAMs results in improved anti-tumoral CD8 T-cell responses and enhanced response to chemotherapy, resulting in prolonged tumor control in a spontaneous MMTV-PyMT murine model of breast cancer. Unfortunately, current HO-1 inhibitors are not orally bioavailable, which limits their utility as immunotherapeutics for the treatment of cancer. In response to this hurdle, we have developed a next generation HO-1 inhibitor called KCL-HO-1i. We demonstrate in pharmacokinetic studies that KCL-HO-1i is orally bioavailable in murine models with a serum half-life of around 3hours. Furthermore, orally delivering KCL-HO-1i alongside standard of care chemotherapy is able to deliver durable tumor control in MMTV-PyMT mice. We have characterized the response of the TME to KCL-HO-1i using flow cytometry, immunofluorescence and RNA sequencing approaches and demonstrate that KCL-HO-1i supports a broader switch from an immunological ‘cold’ to ‘hot’ TME which provides a more favourable immune landscape for improving the response to chemotherapeutic drugs. Taken together, our data support the use of KCL-HO-1i as novel immunotherapeutic for the treatment of cancer. Citation Format: Meriem Bahri, Taha Al-Adhami, Emre Demirel, Joanne E. Anstee, Karen T. Feehan, James Rosekilly, Cheryl E. Gillett, Dominika Sosnowska, Renee Gitsaki-Taylor, Tik Shing Cheung, James F. Spicer, Khondaker Miraz Rahman, James N. Arnold. Targeting perivascular macrophages with an orally bioavailable heme oxygenase-1 inhibitor improves responses to chemotherapeutic drugs in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A013.

Hsa_circ_0129047 Upregulates LYVE1 to Inhibit Hepatocellular Carcinoma Progression by Sponging miR-492.

Compelling evidence indicates the regulatory role of circular RNAs in cancers, including hepatocellular carcinoma (HCC). Our study aimed to elucidate the regulatory function of circ_0129047 in HCC progression. A reverse transcription-quantitative polymeric chain reaction was conducted to detect the expression of circ_0129047, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1), and miR-492 in HCC tissues and cells. The characteristics of circ_0129047 were determined by evaluating the nuclear and cytoplasmic fractions and by RNase R digestion assays. The cell counting kit-8 assay, scratch wound, and transwell invasion assays were used to examine the effects of circ_0129047 overexpression, miR-492 mimic, and LYVE1 overexpression on the proliferation, migration, and invasion abilities of HCC cells in vitro. A mouse xenograft model was also established. The relationship between miR-492 and circ_0129047 or LYVE1 was clarified using luciferase reporter and Argonaute-2 RNA immunoprecipitation assays. We found that circ_0129047 and LYVE1 were poorly expressed in HCC tissues and cells, whereas miR-492 was upregulated. Overexpression of circ_0129047 inhibits HCC cell proliferation, migration, and invasion and delays in vivo tumor growth. Furthermore, circ_0129047 sponged miR-492, and 3'UTR LYVE1 was a direct target of miR-492. Additionally, LYVE1 overexpression reduced the oncogenic activity of the miR-492 mimic, whereas the miR-492 mimic abolished the antimigratory, antiproliferative, and anti-invasive effects of circ_0129047 overexpression in HCC cells. These data suggest that circ_0129047 exerts a tumor-suppressive role in HCC by sponging miR-492 away from LYVE1 and that the circ_0129047/miR-492/LYVE1 axis may be a promising target for HCC treatment.

Three-dimensional visualization of the lymphatic, vascular and neural network in rat lung by confocal microscopy.

In order to demonstrate the intricate interconnection of pulmonary lymphatic vessels, blood vessels, and nerve fibers, the rat lung was selected as the target and sliced at the thickness of 100μm for multiply immunofluorescence staining with lymphatic vessel endothelial hyaluronan receptor 1(LYVE-1), alpha smooth muscle actin (α-SMA), phalloidin, cluster of differentiation 31 (CD31), and protein gene product 9.5 (PGP9.5)antibodies. Taking the advantages of the thicker tissue section and confocal microscopy, the labeled pulmonary lymphatic vessels, blood vessels, and nerve fibers were demonstrated in rather longer distance, which was more convenient to reconstruct a three-dimensional (3D) view for analyzing their spatial correlation in detail. It was clear that LYVE-1+ lymphatic vessels were widely distributed in pulmonary lobules and closely to the lobar bronchus. Through 3D reconstruction, it was also demonstrated that LYVE-1+ lymphatic vessels ran parallel to or around the α-SMA+ venules, phalloidin+ arterioles and CD31+ capillaries, with PGP9.5+ nerve fibers traversing alongside or wrapping around them, forming a lymphatic, vascular and neural network in the lung. By this study, we provide a detailed histological view to highlight the spatial correlation of pulmonary lymphatic, vascular and neural network, which may help us for insight into the functional role of this network under the physiological and pathological conditions.

Widespread distribution of lymphatic vessels in human dura mater remote from sinus veins.

Background and purpose: Previous experimental studies have shown that meningeal lymphatic vessels are located primarily along the walls of the dural sinus veins. Whether they are more widespread throughout human dura mater has presently not been characterized. The present study explored in humans whether meningeal lymphatic vessels may be identified remote from the sinus veins and whether they differ in the various location of dura mater. Methods: We included 15 patients who underwent neurosurgery, in whom dura mater was removed as part of the planned procedure. Tissue was prepared for immunohistochemistry using the lymphatic endothelial cell markers lymphatic vessel endothelial hyaluronan receptor 1 protein (LYVE-1), podoplanin and vascular endothelial growth factor receptor 3 (VEGFR3). Results: Lymphatic endothelial cell positive cells were found in dura mater at the posterior fossa (n = 8), temporal skull base (n = 5), frontal convexity (n = 1), and cranio-cervical junction (n = 1). They were most commonly seen remote from blood vessels, but also occurred along blood vessels, and seemed to be most abundant at the skull base. Conclusion: The present observations show that human lymphatic vessels are widespread in dura mater, not solely lining the dural sinuses.

Angiogenesis-An Emerging Role in Organ Fibrosis.

In recent years, the study of lymphangiogenesis and fibrotic diseases has made considerable achievements, and accumulating evidence indicates that lymphangiogenesis plays a key role in the process of fibrosis in various organs. Although the effects of lymphangiogenesis on fibrosis disease have not been conclusively determined due to different disease models and pathological stages of organ fibrosis, its importance in the development of fibrosis is unquestionable. Therefore, we expounded on the characteristics of lymphangiogenesis in fibrotic diseases from the effects of lymphangiogenesis on fibrosis, the source of lymphatic endothelial cells (LECs), the mechanism of fibrosis-related lymphangiogenesis, and the therapeutic effect of intervening lymphangiogenesis on fibrosis. We found that expansion of LECs or lymphatic networks occurs through original endothelial cell budding or macrophage differentiation into LECs, and the vascular endothelial growth factor C (VEGFC)/vascular endothelial growth factor receptor (VEGFR3) pathway is central in fibrosis-related lymphangiogenesis. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), as a receptor of LECs, is also involved in the regulation of lymphangiogenesis. Intervention with lymphangiogenesis improves fibrosis to some extent. In the complex organ fibrosis microenvironment, a variety of functional cells, inflammatory factors and chemokines synergistically or antagonistically form the complex network involved in fibrosis-related lymphangiogenesis and regulate the progression of fibrosis disease. Further clarifying the formation of a new fibrosis-related lymphangiogenesis network may potentially provide new strategies for the treatment of fibrosis disease.

CD45- erythroid progenitor cells promote lymph node metastasis in gastric cancer by inducing a hybrid epithelial/mesenchymal state in lymphatic endothelial cells.

Specific mechanisms of lymph node (LN) metastasis in early-stage gastric cancer (GC) have not been elucidated. The role of anemia, a vital clinical feature of GC, in LN metastasis is also unclear. Since the number of erythroid progenitor cells (EPCs) is increased in chronic anemia, we investigated its association with LN metastasis in GC. Flow cytometry and immunofluorescence analyses were performed to sort and study EPCs from the circulation and tumors of patients with stage I-III GC. The effect of these EPCs on the activation of T and B cells and on the functions of lymphatic endothelial cells (LECs) was determined, and their ability to promote LN metastasis was evaluated using a footpad-popliteal LN metastasis model based on two human adenocarcinoma GC cell lines in nude mice. The prognostic value of EPCs was also analyzed. The proportion of CD45- EPCs was higher in the mononuclear cells in the circulation, tumors, and LNs of GC patients with LN metastasis (N+) than in those of GC patients without LN metastasis (N0). In N+ patients, CD45- EPCs were more abundant in metastatic LNs than in non-metastatic LNs. Lymphatic vessel endothelial hyaluronan receptor 1 immunoreactivity in tumors revealed that CD45- EPCs were positively associated with nodal stages and lymph vessel density. Furthermore, CD45- EPCs increased LEC proliferation and migration through their S100A8/A9 heterodimer-induced hybrid epithelial/mesenchymal (E/M) state; however, they did not influence the invasion and tubulogenesis of LECs or T and B cell proliferation. CD45- EPCs promoted LN metastasis in vivo; the S100A8/A9 heterodimer mimicked this phenomenon. Finally, CD45- EPCs predicted the overall and disease-free survival of stage I-III GC patients after radical resection. The CD45- EPCs accumulated in GC tissues and metastatic LNs and promoted LN metastasis via the S100A8/9-induced hybrid E/M state of LECs, which was the specific mechanism of LN metastasis in the early stages of GC.

Lymphatic Endothelial-to-Myofibroblast Transition: A Potential New Mechanism Underlying Skin Fibrosis in Systemic Sclerosis.

At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, we herein investigated whether the lymphatic endothelium might represent an additional source of profibrotic myofibroblasts through a lymphatic EndMT (Ly-EndMT) process. Skin sections from patients with SSc and healthy donors were immunostained for the lymphatic endothelial cell-specific marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in combination with α-smooth muscle actin (α-SMA) as the main marker of myofibroblasts. Commercial human adult dermal lymphatic microvascular endothelial cells (HdLy-MVECs) were challenged with recombinant human transforming growth factor-β1 (TGFβ1) or serum from SSc patients and healthy donors. The expression of lymphatic endothelial cell/myofibroblast markers was measured by quantitative real-time PCR, Western blotting and immunofluorescence. Collagen gel contraction assay was performed to assess myofibroblast-like cell contractile ability. Lymphatic endothelial cells in intermediate stages of the Ly-EndMT process (i.e., coexpressing LYVE-1 and α-SMA) were found exclusively in the fibrotic skin of SSc patients. The culturing of HdLy-MVECs with SSc serum or profibrotic TGFβ1 led to the acquisition of a myofibroblast-like morphofunctional phenotype, as well as the downregulation of lymphatic endothelial cell-specific markers and the parallel upregulation of myofibroblast markers. In SSc, the Ly-EndMT might represent a previously overlooked pathogenetic process bridging peripheral microlymphatic dysfunction and skin fibrosis development.

Abstract B001: Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment

Abstract Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive cancer. In the UK, there are around 9000 diagnoses and 3000 deaths annually. Targeted therapies that inhibit angiogenic regulators, such as bevacizumab (monoclonal antibody which inhibits vascular endothelial growth factor A, VEGF-A), have limited efficacy due to drug resistance. Expression of calcitonin receptor-like receptor (CLR) is upregulated in ccRCC and correlates with disease outcome. The expression of adrenomedullin (AM), peptide agonist of CLR and angiogenic factor, is upregulated in ccRCC. We hypothesized that the CLR signaling axis acts as an alternative pathway that enables resistance to drugs targeting the VEGF-A pathway in ccRCC. In the present study, we aimed to determine whether this signaling axis remains active in VEGF-A-stimulated and bevacizumab-treated human endothelial cells. Methods. Primary human dermal blood vessel endothelial cells (HDBEC; PromoCell) were characterized by immunofluorescence utilizing pan-endothelial (cluster of differentiation 31 and 144, CD31 and CD144, and von Willebrand factor, vWF) and lymphatic-specific (lymphatic vessel endothelial hyaluronan receptor 1, LYVE-1, and prospero homeobox 1, PROX1) markers and confocal microscopy. The activities of CLR agonists (AM; intermedin, IMD; calcitonin gene-related peptide, CGRP; 10−12 - 10−6 M at 10 min) and VEGF-A (50 ng/ml over 30-minute time course) were measured by analyzing phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) using immunoblotting. HDBEC were pre-treated with bevacizumab or human IgG1 at 250 ng/ml and then stimulated with individual CLR agonists at 10−6 M for 10 minutes. Experiments were repeated at least three times. Data were analyzed using Shapiro-Wilk normality, unpaired statistics and appropriate multiple comparisons tests utilizing GraphPad Prism and p&amp;lt;0.05 interpreted as significant. Results. HDBEC were a pure population. AM, IMD and CGRP induced p44/42 MAPK phosphorylation at 10−6 M (p&amp;lt;0.05). For all three CLR agonists and VEGF-A, p44/42 MAPK phosphorylation peaked after 10 minutes of incubation (p&amp;lt;0.05). Bevacizumab treatment at 250 ng/ml was sufficient to inhibit VEGF-A-induced p44/42 MAPK phosphorylation (p&amp;lt;0.05). AM, IMD and CGRP were all able to induce phosphorylation of p44/42 MAPK after bevacizumab treatment (p&amp;lt;0.01). Conclusions. The findings that CLR agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated HDBEC after bevacizumab treatment support our hypothesis. This work provides a foundation for investigating the role of the CLR signaling axis in mechanisms of ccRCC resistance to drugs targeting the VEGF-A pathway. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Leonid L. Nikitenko. Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B001.