ERG regulates lymphatic vessel specification genes and its deficiency impairs wound healing-associated lymphangiogenesis.

OBJECTIVE: Rarefaction of blood and lymphatic vessels in the skin has been reported in SSc (systemic sclerosis, scleroderma). ERG and FLI1 are important regulators of angiogenesis, but their role in lymphatic vasculature is less known. The goal of this study was to determine the role of ERG and FLI1 in postnatal lymphangiogenesis and SSc lymphatic system defects. METHODS: Immunofluorescence was used to detect ERG and FLI1 in SSc and healthy control (HC) skin biopsies. Transcriptional analysis of ERG or FLI1 silenced human dermal lymphatic endothelial cells (LECs) was performed using microarrays. Effects of ERG/FLI1 deficiency on in vitro tubulogenesis in human dermal LECs was examined using a Matrigel assay. Erg and Fli1 endothelial specific knockouts and Erg lymphatic specific knockouts were generated to examine vessel regeneration in mice. RESULTS: ERG and FLI1 protein levels were reduced in the blood and lymphatic vasculature in SSc skin biopsies. ERG was shown to regulate genes involved in lymphatic vessel specification, including VEGFR3/FLT4, LYVE-1, SOX18, and PROX1, while FLI1 enhanced the function of ERG. ERG/FLT4 pathway regulated in vitro tubulogenesis in human LECs. Deficiency of Erg or Fli1 similarly impaired the function of blood vessels in mice. However, only Erg deficiency affected the regeneration of lymphatic vessels during wound healing. CONCLUSION: ERG and FLI1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI1 in SSc endothelial cells, may contribute to impairment of blood and lymphatic vasculature in SSc patients.

Endothelial Erg Regulates Expression of Pulmonary Lymphatic Junctional and Inflammation Genes in Mouse Lungs Impacting Lymphatic Transport.

The ETS transcription factor ERG is a master regulator of endothelial gene specificity and highly enriched in the capillary, vein, and arterial endothelial cells. ERG expression is critical for endothelial barrier function, permeability, and vascular inflammation. A dysfunctional vascular endothelial ERG has been shown to impair lung capillary homeostasis, contributing to pulmonary fibrosis as previously observed in IPF lungs. Our preliminary observations indicate that lymphatic endothelial cells (LEC) in the human IPF lung also lack ERG. To understand the role of ERG in pulmonary LECs, we developed LEC-specific inducible Erg-CKO and Erg-GFP-CKO conditional knockout (CKO) mice under Prox1 promoter. Whole lung microarray analysis, flow cytometry, and qPCR confirmed an inflammatory and pro-lymphvasculogenic predisposition in Erg-CKO lung. FITC-Dextran tracing analysis showed an increased pulmonary interstitial lymphatic fluid transport from the lung to the axial lymph node. Single-cell transcriptomics confirmed that genes associated with cell junction integrity were downregulated in Erg-CKO pre-collector and collector LECs. Integrating Single-cell transcriptomics and CellChatDB helped identify LEC specific communication pathways contributing to pulmonary inflammation, trans-endothelial migration, inflammation, and Endo-MT in Erg-CKO lung. Our findings suggest that downregulation of lymphatic Erg crucially affects LEC function, LEC permeability, pulmonary LEC communication pathways and lymphatic transcriptomics.

Increased adipose tissue lymphatic vessel density inhibits thermogenesis through elevated neurotensin levels.

During cold exposure, white adipose tissue can remodel to dissipate energy as heat under cold similar to thermogenic brown adipose tissue. This "browning" and the regulation of body temperature is under the control of neural and hormonal signaling. It was recently discovered that neurotensin, a small neuropeptide, not only acts to inhibit thermogenesis, but also that lymphatic vessels may be a surprisingly potent source of neurotensin production. We hypothesized that the induction of adipose tissue lymphangiogenesis would therefore increase tissue neurotensin levels and impair thermogenesis. Methods: We utilized AdipoVD mice that have inducible expression of vascular endothelial growth factor (VEGF)-D, a potent lymphangiogenic stimulator, specifically in adipose tissue. Overexpression of VEGF-D induced significant lymphangiogenesis in both white and brown adipose tissues of AdipoVD mice. Results: Obese Adipo-VD mice demonstrated no differences in adipose morphology or browning under room temperature conditions compared to controls but did express significantly higher levels of neurotensin in their adipose tissues. Upon acute cold exposure, AdipoVD mice were markedly cold intolerant; inhibition of neurotensin signaling ameliorated this cold intolerance as AdipoVD mice were then able to maintain body temperature on cold challenge equivalent to their littermates. Conclusion: In total, these data demonstrate that adipose tissue lymphatic vessels are a potent paracrine source of neurotensin and that lymphangiogenesis therefore impairs the tissues' thermogenic ability.

Indications of Peripheral Pain, Dermal Hypersensitivity, and Neurogenic Inflammation in Patients with Lipedema.

Lipedema is a disease with abnormally increased adipose tissue deposition and distribution. Pain sensations have been described in the clinical evaluation of lipedema, but its etiology remains poorly understood. We hypothesized that pain sensitivity measurements and ex vivo quantitation of neuronal cell body distribution in the skin would be lipedema stage-dependent, and could, thus, serve to objectively characterize neuropathic pain in lipedema. The pain was assessed by questionnaire and peripheral cutaneous mechanical sensitization (von-Frey) in lipedema (n = 27) and control (n = 23) consenting female volunteers. Dermal biopsies from (n = 11) Stages 1-3 lipedema and control (n = 10) participants were characterized for neuronal cell body and nociceptive neuropeptide calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) distribution. Stage 2 or 3 lipedema participants responded positively to von Frey sensitization in the calf and thigh, and Stage 3 participants also responded in the arm. Lipedema abdominal skin displayed reduced Tuj-1+ neuronal cell body density, compared to healthy controls, while CGRP and NGF was significantly elevated in Stage 3 lipedema tissues. Together, dermal neuronal cell body loss is consistent with hyper-sensitization in patients with lipedema. Further study of neuropathic pain in lipedema may elucidate underlying disease mechanisms and inform lipedema clinical management and treatment impact.

Impact of High Fat Diet and Bolus Feeding on Chyle Accumulation in a Mouse Model of Generalized Lymphatic Anomaly.

Background: Generalized lymphatic anomalies (GLA) are complex vessel malformations that can impair lymphatic function. Potential GLA complications include lipid-rich lymph in the thoracic space or peritoneal cavity, respectively chylothorax and chylous ascites. To reduce the potential for chyle accumulation, GLA patients limit dietary fats. We hypothesized that dietary fatty acid composition impacts the potential for lymphatic dysfunction and chyle accumulation in GLA. Methods and Results: Adipose-specific overexpression of lymphatic growth factors has demonstrated lethal chylothorax in mice. Here, we utilized mice with inducible adipocyte overexpression of vascular endothelial growth factor-D (VD mice) to mimic lymphatic proliferation in GLA and assessed the incidence of chyle accumulation on a mixed high fat diet (HFD), high saturated fat diet (HSFD), or high unsaturated fat diet (HUSFD). Lipid transport was assessed by uptake rates of bolus oral triglyceride load and mesenteric fat analysis. Lymphatic expansion and inflammation were determined by whole mount immunofluorescence and gene expression. Body composition was assessed by MRI. HSFD 2-month wildtype groups resulted in an increase in TNF-α, IL-6, and IL-10 expression compared with chow-fed controls. The chyle accumulation incidence was highest in HFD-fed mice compared with either HSFD or HUSFD. Strikingly, increased mortality was observed irrespective of which high fat diet was consumed after administration of a bolus lipid load. Conclusion: Chronic HFD increases risk of chyle accumulation, however increased mortality was driven particularly by a bolus lipid load in VD mice. These findings suggest that although chronic HFD increases chyle accumulation risk, a single large meal feeding may increase risk of lethal chylothorax instances for GLA patients.

Chronic VEGFR-3 signaling preserves dendritic arborization and sensitization under stress.

Dendritic arborization is critical for the establishment and maintenance of precise neural circuits. Vascular endothelial growth factor D (VEGF-D), well-characterized as a "lymphangiogenic" growth factor, reportedly maintains dendritic arborization and synaptic strength in the hippocampus of adult mice through VEGF receptor (VEGFR-3) signaling. Here, we investigated the effect of chronic VEGFR-3-specific activation on adipose arbor morphometry using the Adipo-VD mouse, a model of inducible, adipose-specific VEGF-D overexpression. We examined whether adipose tissue innervation was preserved or functionally different in Adipo-VD mice during stress in vivo and if VEGFR-3 signaling afforded neuroprotection to challenged neurons in vitro. Chronic VEGFR-3 signaling in Adipo-VD subcutaneous adipose tissue resulted in a reduction in the dendrite length, dendritic terminal branches (filament length), and dendritic terminal branch volume (filament volume), but increased dendrite branching. We also identified reduced stimulus-evoked excitatory sympathetic nerve activity in Adipo-VD mice. Following 6-hydroxydopamine (6-OHDA) denervation, Adipo-VD dendritic arbors were preserved, including improved dendritic branch volume, length, and dendritic branches than in wildtype tissues. In vitro, we found that chronic elevation of VEGFR-3 signaling in developing mVC neurons changes the dendritic arbor complexity and improves stress-induced structure remodeling. Developing neurons are conferred neuroprotection against stress, potentially by upregulation of proteolytic conversion of pro-BDNF to mature BDNF. Mature neurons, however, display improved dendritic arbor complexity, and unaltered dendritic structural remodeling and improved resistance to stress with VEGFR-3 signaling. Overall, chronically increasing VEGFR-3 signaling in neurons has a synergistic impact on neurosensitization and neuroprotection during stress.

Characterizing Lymphangiogenesis and Concurrent Inflammation in Adipose Tissue in Response to VEGF-D.

The metabolic consequences of obesity arise from local inflammation within expanding adipose tissue. In pre-clinical studies targeting various inflammatory factors, systemic metabolism can be improved through reduced adipose inflammation. Lymphatic vessels are a critical regulator of inflammation through roles in fluid and macromolecule transport and immune cell trafficking and immunomodulation. Lymphangiogenesis, the expansion of the lymphatic network, is often a necessary step in restoring tissue homeostasis. Using Adipo-VD mice, a model of adipocyte-specific, inducible overexpression of the potent lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D), we previously identified that dense de novo adipose lymphatics reduced immune accumulation and improved glucose homeostasis in obesity. On chow diet, however, Adipo-VD mice demonstrated increased adipose tissue immune cells, fibrosis, and inflammation. Here, we characterize the time course of resident macrophage accumulation and lymphangiogenesis in male and female Adipo-VD mice fed chow and high fat diets, examining multiple adipose depots over 4 months. We find that macrophage infiltration occurs early, but resolves with concurrent lymphatic expansion that begins robustly after 1 month of VEGF-D overexpression in white adipose tissue. In obesity, female Adipo-VD mice exhibit reduced lymphangiogenesis and maintain a more glycolytic metabolism compared to Adipo-VD males and their littermates. Adipose lymphatic structures appear to expand by a lymphvasculogenic mechanism involving lymphatic endothelial cell proliferation and organization with a cell source we that failed to identify; hematopoietic cells afford minimal structural contribution. While a net positive effect occurs in Adipo-VD mice, adipose tissue lymphangiogenesis demonstrates a dichotomous, and time-dependent, inflammatory tissue remodeling response.

Vascular Endothelial Growth Factor-D (VEGF-D) Overexpression and Lymphatic Expansion in Murine Adipose Tissue Improves Metabolism in Obesity.

Obese adipose tissue expansion is an inflammatory process that results in dysregulated lipolysis, increased circulating lipids, ectopic lipid deposition, and systemic insulin resistance. Lymphatic vessels provide a route of fluid, macromolecule, and immune cell clearance, and lymphangiogenesis increases this capability. Indeed, inflammation-associated lymphangiogenesis is critical in resolving acute and chronic inflammation, but it is largely absent in obese adipose tissue. Enhancing adipose tissue lymphangiogenesis could, therefore, improve metabolism in obesity. To test this hypothesis, transgenic mice with doxycycline-inducible expression of murine vascular endothelial growth factor (VEGF)-D under a tightly controlled Tet-On promoter were crossed with adipocyte-specific adiponectin-reverse tetracycline-dependent transactivator mice (Adipo-VD) to stimulate adipose tissue-specific lymphangiogenesis during 16-week high-fat diet-induced obesity. Adipose VEGF-D overexpression induced de novo lymphangiogenesis in murine adipose tissue, and obese Adipo-VD mice exhibited enhanced glucose clearance, lower insulin levels, and reduced liver triglycerides. On ß-3 adrenergic stimulation, Adipo-VD mice exhibited more rapid and increased glycerol flux from adipose tissue, suggesting that the lymphatics are a potential route of glycerol clearance. Resident macrophage crown-like structures were scarce and total F4/80+ macrophages were reduced in obese Adipo-VD s.c. adipose tissue with evidence of increased immune trafficking from the tissue. Augmenting VEGF-D signaling and lymphangiogenesis specifically in adipose tissue, therefore, reduces obesity-associated immune accumulation and improves metabolic responsiveness.

The Role of NMDA Receptors in Neural Stem Cell Proliferation and Differentiation.

N-methyl-d-aspartate receptors (NMDARs) are expressed abundantly in the brain and play an important role in the regulation of neuronal development, learning, memory, neurodegenerative diseases, and neurogenesis, all of which are extensively discussed in reviews published elsewhere. While the number of research articles on the developmental and neurological cues that trigger NMDAR localization across a developing neuron and the role of the NMDAR in embryonic and adult stem cell proliferation and differentiation have been growing dramatically over the past few years, there is not a single review on this latter general topic. In the proposed review, we will summarize the current understanding of NMDARs in stem cell biology and their involvement in pathophysiological processes of neuron development, especially during early neuronal development (immature neurons) and differentiation.