Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.

During development, the lymphatic vasculature forms as a second network derived chiefly from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is a key step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet the downstream mechanisms remain to be elucidated. We here present a single-cell transcriptomic atlas of lymphangiogenesis in zebrafish, revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single-cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a LEC-VEC fate shift. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new downstream targets and regulatory regions of the genome controlled by Prox1 and presents evidence that Prox1 specifies LEC fate primarily by limiting blood vascular and haematopoietic fate. This extensive single-cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

The blood vasculature instructs lymphatic patterning in a SOX7-dependent manner.

Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.

Vascular endothelial growth factor c regulates hematopoietic stem cell fate in the dorsal aorta.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells that self-renew or differentiate to establish the entire blood hierarchy. HSPCs arise from the hemogenic endothelium of the dorsal aorta (DA) during development in a process called endothelial-to-hematopoietic transition. The factors and signals that control HSPC fate decisions from the hemogenic endothelium are not fully understood. We found that Vegfc has a role in HSPC emergence from the zebrafish DA. Using time-lapse live imaging, we show that some HSPCs in the DA of vegfc loss-of-function embryos display altered cellular behavior. Instead of typical budding from the DA, emergent HSPCs exhibit crawling behavior similar to myeloid cells. This was confirmed by increased myeloid cell marker expression in the ventral wall of the DA and the caudal hematopoietic tissue. This increase in myeloid cells corresponded with a decrease in HSPCs that persisted into larval stages. Together, our data suggest that Vegfc regulates HSPC emergence in the hemogenic endothelium, in part by suppressing a myeloid cell fate. Our study provides a potential signal for modulation of HSPC fate in stem cell differentiation protocols.

The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafish.

BACKGROUND: Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. RESULTS: We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. CONCLUSIONS: Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.

Lymphangiogenesis guidance by paracrine and pericellular factors.

Lymphatic vessels are important for tissue fluid homeostasis, lipid absorption, and immune cell trafficking and are involved in the pathogenesis of several human diseases. The mechanisms by which the lymphatic vasculature network is formed, remodeled, and adapted to physiological and pathological challenges are controlled by an intricate balance of growth factor and biomechanical cues. These transduce signals for the readjustment of gene expression and lymphatic endothelial migration, proliferation, and differentiation. In this review, we describe several of these cues and how they are integrated for the generation of functional lymphatic vessel networks.

The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

The secreted protein collagen and calcium-binding EGF domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFß-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in patients with CRC. YAP/TAZ-TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts, which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells in vitro and lymphangiogenesis in a CRC cell-derived xenograft model in vivo. In addition, the bromodomain and extraterminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis, and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ-TEAD4-BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.

Enhanced meningeal lymphatic drainage ameliorates lipopolysaccharide-induced brain injury in aged mice.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is an acute cerebral dysfunction caused by sepsis. Neuroinflammation induced by sepsis is considered a potential mechanism of SAE; however, very little is known about the role of the meningeal lymphatic system in SAE. METHODS: Sepsis was established in male C57BL/6J mice by intraperitoneal injection of 5 mg/kg lipopolysaccharide, and the function of meningeal lymphatic drainage was assessed. Adeno-associated virus 1-vascular endothelial growth factor C (AAV1-VEGF-C) was injected into the cisterna magna to induce meningeal lymphangiogenesis. Ligation of deep cervical lymph nodes (dCLNs) was performed to induce pre-existing meningeal lymphatic dysfunction. Cognitive function was evaluated by a fear conditioning test, and inflammatory factors were detected by enzyme-linked immunosorbent assay. RESULTS: The aged mice with SAE showed a significant decrease in the drainage of OVA-647 into the dCLNs and the coverage of the Lyve-1 in the meningeal lymphatic, indicating that sepsis impaired meningeal lymphatic drainage and morphology. The meningeal lymphatic function of aged mice was more vulnerable to sepsis in comparison to young mice. Sepsis also decreased the protein levels of caspase-3 and PSD95, which was accompanied by reductions in the activity of hippocampal neurons. Microglia were significantly activated in the hippocampus of SAE mice, which was accompanied by an increase in neuroinflammation, as indicated by increases in interleukin-1 beta, interleukin-6 and Iba1 expression. Cognitive function was impaired in aged mice with SAE. However, the injection of AAV1-VEGF-C significantly increased coverage in the lymphatic system and tracer dye uptake in dCLNs, suggesting that AAV1-VEGF-C promotes meningeal lymphangiogenesis and drainage. Furthermore, AAV1-VEGF-C reduced microglial activation and neuroinflammation and improved cognitive dysfunction. Improvement of meningeal lymphatics also reduced sepsis-induced expression of disease-associated genes in aged mice. Pre-existing lymphatic dysfunction by ligating bilateral dCLNs aggravated sepsis-induced neuroinflammation and cognitive impairment. CONCLUSION: The meningeal lymphatic drainage is damaged in sepsis, and pre-existing defects in this drainage system exacerbate SAE-induced neuroinflammation and cognitive dysfunction. Promoting meningeal lymphatic drainage improves SAE. Manipulation of meningeal lymphangiogenesis could be a new strategy for the treatment of SAE.

Examining the correlation of lymphangiogenesis biomarkers with clinical condition in Age-Related Macular Degeneration (AMD).

The aim of this study is to investigate the relationship between age-related macular degeneration (AMD) and lymphangiogenesis biomarkers, namely LYVE-1, Podoplanin, VEGF-C, VEGFR-2 and VEGFR-3. This prospective and interventional study includes 30 patients with AMD which may be dry or wet type and 30 controls for whom vitrectomy and phacoemulsification was indicated due to additional pathologies (epiretinal membrane, macular hole, retinal detachment, and cataract). 0.1-0,2 ml of aqueous humor and 0.5-1 ml of vitreous sample was taken during the operations. Before the operations 1 tube serum was also taken. All the lymphangiogenesis biomarkers in the study are examined by ELISA method. LYVE-1 (p = 0.001) and Podoplanin (p = 0.004) levels in the vitreous for the patient group are found to be significantly lower than the control group. Serum (p = 0.019), vitreous (p = 0.001), aqueous (p < 0.001) levels of VEGF-C for the patient group are significantly higher than the control group. VEGF-C/VEGFR-2 (p < 0.001), VEGF-C/VEGFR-3 (p < 0.001) ratios in the vitreous for the patient group are found to be significantly higher than the control group. Especially in wet AMD patients, LYVE-1 level is significantly lower in the vitreous (p = 0.002) and aqueous (p = 0.002) than the control group. In addition, Podoplanin level is observed as significantly lower in the vitreous (p = 0.014) and serum (p = 0.002) in comparison to control group. In the wet AMD group, VEGF-C level in the vitreous (p < 0.001), aqueous (p < 0.001) and serum (p = 0.001) is higher than the control group. The result of this study indicates a valid relationship between the weakening of lymphangiogenesis and the pathophysiology of AMD, especially for the wet type. It is observed that the levels of receptors that bind VEGF-C (VEGFR-2 and VEGFR-3) do not increase at the same rate as VEGF-C to compensate for the increase in VEGF-C. The absence of an increase in VEGFR-3, which is especially necessary for lymphangiogenesis, also suggests that lymphangiogenesis is weakened or decreased in AMD. In the future interventional studies with larger series, examination of lymphangiogenic biomarkers in inflammatory retinal diseases and glaucoma may reveal unexplored details.

VEGFC ameliorates salt-sensitive hypertension and hypertensive nephropathy by inhibiting NLRP3 inflammasome via activating VEGFR3-AMPK dependent autophagy pathway.

Salt-sensitivity hypertension (SSHTN) is an independent predictor for cardiovascular mortality. VEGFC has been reported to be a protective role in SSHTN and hypertensive kidney injury. However, the underlying mechanisms remain largely unclear. The current study aimed to explore the protective effects and mechanisms of VEGFC against SSHTN and hypertensive nephropathy. Here, we reported that VEGFC attenuated high blood pressure as well as protected against renal inflammation and fibrosis in SSHTN mice. Moreover, VEGFC suppressed the activation of renal NLRP3 inflammasome in SSHTN mice. In vitro, we found VEGFC inhibited NLRP3 inflammasome activation, meanwhile, upregulated autophagy in high-salt-induced macrophages, while these effects were reversed by an autophagy inhibitor 3MA. Furthermore, in vivo, 3MA pretreatment weakened the protective effects of VEGFC on SSHTN and hypertensive nephropathy. Mechanistically, VEGF receptor 3 (VEGFR3) kinase domain activated AMPK by promoting the phosphorylation at Thr183 via binding to AMPK, thus enhancing autophagy activity in the context of high-salt-induced macrophages. These findings indicated that VEGFC inhibited NLRP3 inflammasome activation by promoting VEGFR3-AMPK-dependent autophagy pathway in high-salt-induced macrophages, which provided a mechanistic basis for the therapeutic target in SSHTN and hypertensive kidney injury.

Understanding the development, pathogenesis, and injury response of meningeal lymphatic networks through the use of animal models.

Meningeal lymphatic vessels (MLVs) help maintain central nervous system (CNS) homeostasis via their ability to facilitate macromolecule waste clearance and neuroimmune trafficking. Although these vessels were overlooked for centuries, they have now been characterized in humans, non-human primates, and rodents. Recent studies in mice have explored the stereotyped growth and expansion of MLVs in dura mater, the various transcriptional, signaling, and environmental factors regulating their development and long-term maintenance, and the pathological changes these vessels undergo in injury, disease, or with aging. Key insights gained from these studies have also been leveraged to develop therapeutic approaches that help augment or restore MLV functions to improve brain health and cognition. Here, we review fundamental processes that control the development of peripheral lymphatic networks and how these might apply to the growth and expansion of MLVs in their unique meningeal environment. We also emphasize key findings in injury and disease models that may reveal additional insights into the plasticity of these vessels throughout the lifespan. Finally, we highlight unanswered questions and future areas of study that can further reveal the exciting therapeutic potential of meningeal lymphatics.

miR-455-3p regulates lymphangiogenesis in silicosis by regulating VEGF-C/VEGFR3.

Silicosis is a disease characterized by lung inflammation and fibrosis caused by long-term inhalation of free silicon dioxide (SiO2). Recent studies have found that a large number of lymphatic hyperplasia occurs during the occurrence and development of silicosis. miRNAs play an important role in lymphangiogenesis. However, the regulation and mechanism of miRNAs on lymphangiogenesis in silicosis remain unclear. In this study, lymphangiogenesis was observed in silicosis rats, and VEGF-C-targeted miRNAs were screened, and the effect of miRNAs on the formation of human lymphatic endothelial cells (HLECs) tubular structure was investigated in vitro. The results showed that SiO2 promoted the expressions of Collagen Ι and α-SMA, TNF-α, IL-6 and VEGF-C increased first and then decreased, and promoted the formation of lymphatic vessels. Bioinformatics methods screened miR-455-3p for targeted binding to VEGF-C, and dual luciferase reporter genes confirmed VEGF-C as the target gene of miR-455-3p, and miR-455-3p was down-regulated in the lung tissue of silicosis rats. Transfection of miR-455-3p Inhibitors down-regulated the expression level of miR-455-3p and up-regulated the expression levels of VEGF-C and VEGFR-3 in HLECs, enhanced migration ability and increased tube formation. Transfection of miR-455-3p Mimics showed an opposite trend. These results suggest that miR-455-3p further regulates the tubular structure formation of HLECs by regulating VEGF-C/VEGFR3. Therefore, targeting miR-455-3p may provide a new therapeutic strategy for SiO2-induced silicosis injury.

Role of CD44-Positive Extracellular Vesicles Derived from Highly Metastatic Mouse Mammary Carcinoma Cells in Pre-Metastatic Niche Formation.

Malignant breast cancers pose a notable challenge when it comes to treatment options. Recently, research has implicated extracellular vesicles (EVs) secreted by cancer cells in the formation of a pre-metastatic niche. Small clumps of CD44-positive breast cancer cells are efficiently transferred through CD44-CD44 protein homophilic interaction. This study aims to examine the function of CD44-positive EVs in pre-metastatic niche formation in vitro and to suggest a more efficacious EV formulation. We used mouse mammary carcinoma cells, BJMC3879 Luc2 (Luc2 cells) as the source of CD44-positive EVs and mouse endothelial cells (UV2 cells) as the recipient cells in the niche. Luc2 cells exhibited an enhanced secretion of EVs expressing CD44 and endothelial growth factors (VEGF-A, -C) under 20% O2 (representative of the early stage of tumorigenesis) compared to its expression under 1% O2 (in solid tumor), indicating that pre-metastatic niche formation occurs in the early stage. Furthermore, UV2 endothelial cells expressing CD44 demonstrated a high level of engulfment of EVs that had been supplemented with hyaluronan, and the proliferation of UV2 cells occurred following the engulfment of EVs. These results suggest that anti-VEGF-A and -C encapsulated, CD44-expressing, and hyaluronan-coated EVs are more effective for tumor metastasis.

Expansion and collapse of VEGF diversity in major clades of the animal kingdom.

Together with the platelet-derived growth factors (PDGFs), the vascular endothelial growth factors (VEGFs) form the PDGF/VEGF subgroup among cystine knot growth factors. The evolutionary relationships within this subgroup have not been examined thoroughly to date. Here, we comprehensively analyze the PDGF/VEGF growth factors throughout all animal phyla and propose a phylogenetic tree. Vertebrate whole-genome duplications play a role in expanding PDGF/VEGF diversity, but several limited duplications are necessary to account for the temporal pattern of emergence. The phylogenetically oldest PDGF/VEGF-like growth factor likely featured a C-terminus with a BR3P signature, a hallmark of the modern-day lymphangiogenic growth factors VEGF-C and VEGF-D. Some younger VEGF genes, such as VEGFB and PGF, appeared completely absent in important vertebrate clades such as birds and amphibia, respectively. In contrast, individual PDGF/VEGF gene duplications frequently occurred in fish on top of the known fish-specific whole-genome duplications. The lack of precise counterparts for human genes poses limitations but also offers opportunities for research using organisms that diverge considerably from humans. Sources for the graphical abstract: 326 MYA and older [1]; 72-240 MYA [2]; 235-65 MYA [3].

Lymphatic Vasculature: An Emerging Therapeutic Target and Drug Delivery Route.

The lymphatic system has received increasing scientific and clinical attention because a wide variety of diseases are linked to lymphatic pathologies and because the lymphatic system serves as an ideal conduit for drug delivery. Lymphatic vessels exert heterogeneous roles in different organs and vascular beds, and consequently, their dysfunction leads to distinct organ-specific outcomes. Although studies in animal model systems have led to the identification of crucial lymphatic genes with potential therapeutic benefit, effective lymphatic-targeted therapeutics are currently lacking for human lymphatic pathological conditions. Here, we focus on the therapeutic roles of lymphatic vessels in diseases and summarize the promising therapeutic targets for modulating lymphangiogenesis or lymphatic function in preclinical or clinical settings. We also discuss considerations for drug delivery or targeting of lymphatic vessels for treatment of lymphatic-related diseases. The lymphatic vasculature is rapidly emerging as a critical system for targeted modulation of its function and as a vehicle for innovative drug delivery.

Molecular mechanisms of miR-1236 in the assessment of tumor lymphangiogenesis in human ovarian cancer patients.

BACKGROUND: Tumor lymphangiogenesis is a critical component in the progression of cancers and specific microRNAs have been reported to be implicated in this process. Recent studies revealed the involvement of miR-1236 in lymphangiogenic signaling by targeting vascular endothelial growth factor receptor 3 (VEGFR3). However, the prognostic importance of miR-1236 and its clinical relevance for lymphangiogenesis in ovarian cancer (OC) remains unclear. METHODS: The study included 52 ovarian tumors and 28 normal ovarian tissues. Quantitative real-time PCR was utilized to analyze the VEGFR3, VEGF-C, LYVE-1 and PROX1 mRNA expression as well as miR-1236. VEGFR3 protein expression was measured by immunohistochemistry staining. Immunohistochemistry for the podoplanin marker (D2-40) was performed to measure lymphatic vessel density (LVD). In addition, diagnostic evaluation based on the receiver-operating characteristic (ROC) curve was performed. The influence of miR-1236 on overall survival was evaluated by Kaplan-Meier method. RESULTS: Here, we show that miR-1236 expression was significantly decreased in ovarian tumors compared with control tissues (p < 0.001) and correlated with advanced clinical stage, lymph node metastasis, distant metastasis and patient survival (All P < 0.05). Moreover, in ovarian tumors, LVD as well as the gene expression of VEGFR3, VEGF-C and LYVE-1, but not PROX1, were found to be remarkably higher compared with control tissues. We also detected a more robust positive staining for VEGFR3 in OC tissues than in control tissues. Furthermore, our results demonstrated an inverse association of miR-1236 expression with LVD, VEGFR3, LYVE-1 and PROX1 expression in OC tissues. The ROC curve analysis indicated that miR-1236 expression has the potential to be used as a diagnostic and prognostic biomarker in OC. Survival analysis further verified a lowered overall survival rate in patients with low miR-1236 expression than in those with high expression. CONCLUSIONS: Our results provide evidence for the translational involvement of miR-1236 in the lymphangiogenesis of OC by regulating lymphangiogenesis-related factors and support the clinical importance of miR-1236 as a new diagnostic and prognostic biomarker for OC.

Lymphatics in bone arise from pre-existing lymphatics.

Bones do not normally have lymphatics. However, individuals with generalized lymphatic anomaly (GLA) or Gorham-Stout disease (GSD) develop ectopic lymphatics in bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known and the development of bone lymphatics has not been fully characterized. Here, we describe the development of bone lymphatics in mouse models of GLA and GSD. Through lineage-tracing experiments, we show that bLECs arise from pre-existing Prox1-positive LECs. We show that bone lymphatics develop in a stepwise manner where regional lymphatics grow, breach the periosteum and then invade bone. We also show that the development of bone lymphatics is impaired in mice that lack osteoclasts. Last, we show that rapamycin can suppress the growth of bone lymphatics in our models of GLA and GSD. In summary, we show that bLECs can arise from pre-existing LECs and that rapamycin can prevent the growth of bone lymphatics.

YAP and TAZ maintain PROX1 expression in the developing lymphatic and lymphovenous valves in response to VEGF-C signaling.

Lymphatic vasculature is an integral part of digestive, immune and circulatory systems. The homeobox transcription factor PROX1 is necessary for the development of lymphatic vessels, lymphatic valves (LVs) and lymphovenous valves (LVVs). We and others previously reported a feedback loop between PROX1 and vascular endothelial growth factor-C (VEGF-C) signaling. PROX1 promotes the expression of the VEGF-C receptor VEGFR3 in lymphatic endothelial cells (LECs). In turn, VEGF-C signaling maintains PROX1 expression in LECs. However, the mechanisms of PROX1/VEGF-C feedback loop remain poorly understood. Whether VEGF-C signaling is necessary for LV and LVV development is also unknown. Here, we report for the first time that VEGF-C signaling is necessary for valve morphogenesis. We have also discovered that the transcriptional co-activators YAP and TAZ are required to maintain PROX1 expression in LVs and LVVs in response to VEGF-C signaling. Deletion of Yap and Taz in the lymphatic vasculature of mouse embryos did not affect the formation of LVs or LVVs, but resulted in the degeneration of these structures. Our results have identified VEGF-C, YAP and TAZ as a crucial molecular pathway in valve development.

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

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

Relationship of Vascular Endothelial Growth Factor C, a Lymphangiogenesis Modulator, With Edema Formation, Congestion and Outcomes in Acute Heart Failure.

BACKGROUND: Although vascular endothelial growth factor C (VEGF-C) is a known lymphangiogenesis modulator, its relationship with congestion formation and outcomes in acute heart failure (AHF) is unknown. METHODS: Serum VEGF-C levels were measured in 237 patients hospitalized for AHF. The population was stratified by VEGF-C levels and linked with clinical signs of congestion and outcomes. RESULTS: The study's population was divided in VEGF-C tertiles: low (median [Q25-Q75]: 33 [15-175]), medium (606 [468-741]) and high (1141 [968-1442] pg/mL). The group with low VEGF-C on admission presented with the highest prevalence of severe lower-extremity edema (low VEGF-C vs medium VEGF-C vs high VEGF-C): 30% vs 13% vs 20%; P = 0.02); the highest percentage of patients with ascites: 22% vs 9% vs 6%; P = 0.006; and the lowest proportion of patients with pulmonary congestion: 22% vs 30% vs 46%; P = 0.004. The 1-year mortality rate was the highest in the low VEGF-C tertile: 35% vs 28% vs 18%, respectively; P = 0.049. The same pattern was observed for the composite endpoint (death and AHF rehospitalization): 45% vs 43% vs 26%; P = 0.029. The risks of death at 1-year follow-up and composite endpoint were significantly lower in the high VEGF-C group. CONCLUSIONS: Low VEGF-C was associated with more severe signs of congestion (signs of fluid accumulation) and adverse clinical outcomes.

Plasminogen activating inhibitor-1 promotes angiogenesis in cutaneous angiosarcomas.

Plasminogen activating inhibitor-1 (PAI-1) is associated with poor clinical outcomes, and elevated levels of PAI-1 in both tissue and serum are correlated with poor response to therapy in various cancers, including skin cancer. Cutaneous angiosarcoma (CAS) is a vascular tumor histologically characterized by detachment of endothelial cell-derived tumor cells. Since CAS expresses multiple angiogenic growth factors and has increased expressions of angiogenic receptor tyrosine kinase transcripts including VEGFR1/2/3, angiogenesis-promoting factors are potential drug targets in CAS. In this study, the expression of PAI-1 was examined in 31 cases of CAS, and the immunomodulatory effects of PAI-1 on a human CAS cell line, ISO-HAS-B, were evaluated. We found that, of the angiogenesis-promoting factors, PAI-1 was expressed in almost all cases of CAS, and PAI-1 increased the mRNA expressions of IL-23p19, VEGF-C, CXCL5 and CCL20 on ISO-HAS-B. Moreover, PAI-1 stimulated ISO-HAS-B culture supernatant promoted favourable tube networks, suggesting that these tumor-derived factors promote the pro-angiogenic effect on tumor development. In addition, IL-23p19 was expressed in 61.3% of cases, whereas VEGF-C was expressed in 41% of cases. The results of the present study suggest that PAI-1 promotes angiogenesis that results in tumor progression in CAS.