Factor Forkhead Box C1 Research Articles

Pharmacogenetic association of the NR1H3 promoter variant with antihypertensive response among patients with hypertension: A longitudinal study.

Background: The genetic factors in assessing therapeutic efficacy and predicting antihypertensive drug response are unclear. Therefore, this study aims to identify the associations between variants and antihypertensive drug response. Methods: A longitudinal study including 1837 hypertensive patients was conducted in Northern China and followed up for a median 2.24years. The associations of 11 candidate variants with blood pressure changes in response to antihypertensive drugs and with the risk of cardiovascular events during the follow-up were examined. The dual-luciferase assay was carried out to assess the effect of genetic variants on gene transcriptional activity. Results: The variant rs11039149A>G in the promoter of nuclear receptor subfamily 1 group H member 3 (NR1H3) was associated with the change in systolic blood pressure (ΔSBP) in response to calcium channel blockers (CCBs) monotherapy. Patients carrying rs11039149AG genotype showed a significant increase of systolic blood pressure (SBP) at follow-up compared with AA carriers, and the difference of ΔSBP between AG and AA carriers was 5.94mm Hg (95%CI: 2.09-9.78, p = 0.002). In 1,184 patients with CCBs therapy, SBP levels decreased in AA carriers, but increased in AG carriers, the difference of ΔSBP between AG and AA carriers was 8.04mm Hg (95%CI: 3.28-12.81, p = 0.001). Further analysis in 359 patients with CCBs monotherapy, the difference of ΔSBP between AG and AA carriers was 15.25mm Hg (95%CI: 6.48-24.02, p = 0.001). However, there was no significant difference in ΔSBP between AG and AA carriers with CCBs multitherapy. The rs11039149A>G was not associated with the cardiovascular events incidence during the follow-up. Additionally, transcriptional factor forkhead box C1 (FOXC1) bound to the NR1H3 promoter containing rs11039149A and significantly increased the transcriptional activity, while rs11039149 A to G change led to a loss-of-function and disabled FOXC1 binding. For the other 10 variants, associations with blood pressure changes or risk of cardiovascular events were not observed. Conclusion: Hypertensive patients with rs11039149AG genotype in the NR1H3 gene have a significant worse SBP control in response to CCBs monotherapy compared with AA carriers. Our findings suggest that the NR1H3 gene might act as a promising genetic factor to affect individual sensitivity to antihypertensive drugs.

Abstract 222: The Molecular Mechanisms Of Coronary Artery Disease Risk At The Pdgfd Locus

Background: Atherosclerotic cardiovascular diseases, including coronary artery disease (CAD), are the worldwide leading cause of death. Recent genome wide association studies (GWAS) have now identified over 250 genetic loci that are associated with CAD, and efforts in this lab and others are investigating the mechanisms of association at these loci. The CAD GWAS locus at 11q22.3 is of potential interest because it encodes the PDGFD gene, a member of the platelet-derived growth factor family whose expression is up-regulated in atherosclerosis. But whether disease allelic variation regulates PDGFD expression and how this growth factor might influence the risk of CAD has not been studied. Methods: The causal variant and gene were investigated with colocalization and in vitro transcription studies in cultured human coronary artery smooth muscle cells (SMC). Single-cell transcriptomic profiling (scRNAseq) and histological analyses were conducted in SMC-specific lineage tracing in an in vivo Pdgfd knockout mouse model of atherosclerosis. Results: The causal variant rs2019090 was linked to PDGFD expression in SMC. In silico analysis of transcription factor binding site prediction and in vitro haplotype-specific chromatin immunoprecipitation combined with qPCR analysis revealed another causal CAD factor forkhead box c1 (FOXC1) preferentially binds to the A-allele of variant rs2019090 and controls the expression level of PDGFD . scRNAseq data identified that loss of Pdgfd resulted in a marked increase in differentiated SMC but a decrease in fibromyocyte (FMC) and chondromyocyte (CMC) transition populations, as well as a significant decrease in plaque macrophages. Differentially expressed gene analysis in SMC lineage cells was highly enriched for CMC genes such as Col2a1 and Ibsp. Lesion histology analysis identified an increase in medial SMC, but a decrease in fibrous cap SMC, with decreased Cd68 and Col2a1 stained area. Conclusion: PDGFD is a causal CAD gene that increases disease risk by promoting SMC transition primarily to the CMC phenotype, and recruitment of monocyte-macrophage lineage cells to disease lesions. These findings support suppression of PDGFD as a possible therapy for human atherosclerotic disease.

Single-gene diagnostic assay for rapid subclassification of basal like breast cancer with mRNA targeted antisense oligonucleotide capped molecular probe

Basal-like breast cancers (BLBCs) represent a subset of ∼70% of triple negative breast cancers (TNBCs) and is significantly more aggressive than tumors of other molecular subtypes. The primary hurdle to develop an effective therapeutic possibility for BLBC lies in the failure to accurately classify this molecular subtype using typical histopathological practices. A clear clinical unmet need is therefore to develop a simple robust diagnostic assay for BLBC that can be made widely available to every pathology laboratory. Elevated expression of transcription factor Forkhead box C1 (FOXC1) has recently been reported as an important prognostic biomarker and functional regulator of BLBC. We herein developed a new nanotechnology-enabled molecular diagnostic assay that will target FOXC1 to directly classify BLBC types from tissue extracts. The molecular assay is comprised of unique antisense oligonucleotide (ASO) guided nanoprobe designed for specific targeting of FOXC1 mRNA. The developed assay has been optimized and validated analytically and demonstrated for its clinical applicability in a small cohort of breast cancer tissues. The significance of the results was further established and corroborated using FOXC1 protein-based ELISA, RT-PCR and fluorescence in-situ hybridization (FISH) assay by detecting the endogenous mRNAs at the ultrastructural level. The results reported herein could greatly improve accessibility of single gene based oncologic assay and help accelerate the accurate identification of this aggressive disease.

RETRACTED: The Sp1/FOXC1/HOTTIP/LATS2/YAP/β‐catenin cascade promotes malignant and metastatic progression of osteosarcoma

The prognosis for osteosarcoma (OS) is dismal due to the aggressive tumor growth and high incidence of metastasis. The long noncoding RNA human homeobox A transcript at the distal tip (HOTTIP) and the transcription factor forkhead box C1 (FOXC1) present oncogenic activities in OS. Here, we aimed at gaining insights into the underlying mechanisms and their crosstalk. The expression of FOXC1 and HOTTIP in OS tissues or cell lines was examined by real-time PCR (RT-PCR) and western blot. The invitro effects of FOXC1 or HOTTIP on cell viability, proliferation, migration, invasion, and expression of target genes were examined using MTT, colony-forming assay, wound-healing, Transwell invasion, and western blot, respectively; the invivo effects were examined using xenograft and experimental metastasis models. Molecular control of HOTTIP on large tumor suppressor 2 (LATS2) or transactivation of FOXC1 or Sp1 on HOTTIP was assessed by combining RNA immunoprecipitation, qRT-PCR, western blot, ChIP, and luciferase assay. Both FOXC1 and HOTTIP were potently up-regulated in OS tissues and cell lines. FOXC1 and HOTTIP essentially maintained viability, proliferation, migration, and invasion of OS cells invitro and contributed to xenograft growth or lung metastasis invivo. Mechanistically, HOTTIP recruited enhancer of zeste homolog 2 (EZH2) and lysine-specific demethylase 1 (LSD1) to silence LATS2 and thus activated YAP/β-catenin signaling. Upstream, Sp1 activated FOXC1 and they both directly transactivated HOTTIP. In summary, we showed that the Sp1/FOXC1/HOTTIP/LATS2/YAP/β-catenin cascade presented oncogenic activities in OS cells. Targeting FOXC1 or HOTTIP may therefore prove beneficial for OS treatment.

Leukemia cells remodel marrow adipocytes via TRPV4-dependent lipolysis.

Remodeling of adipocyte morphology and function plays a critical role in prostate cancer development. We previously reported that leukemia cells secrete growth differentiation factor 15 (GDF15), which remodels the residual bone marrow (BM) adipocytes into small adipocytes and is associated with a poor prognosis in patients with acute myeloid leukemia. However, little is known about how GDF15 drives BM adipocyte remodeling. In this study, we examined the role of the transient receptor potential vanilloid (TRPV) channels in the remodeling of BM adipocytes exposed to GDF15. We found that TRPV4 negatively regulated GDF15-induced remodeling of BM adipocytes. Furthermore, transforming growth factor-β type II receptor was identified as the main receptor for GDF15 on BM adipocytes. PI3K inhibitor treatment reduced GDF15- induced pAKT, identifying PI3K/AKT as the downstream stress response pathway. Subsequently, GDF15 reduced the expression of the transcription factor Forkhead box C1 (FOXC1) in BM adipocytes subjected to RNAsequencing screening and western blot analysis. Moreover, it was also confirmed that FOXC1 combined with the TRPV4 promoter by chromatin immunoprecipitation with quantitative polymerase chain reaction experiments, which suggests that FOXC1 mediates GDF15 regulation of TRPV4. In addition, an acute myeloid leukemia mouse model exhibited smaller BM adipocytes, whereas the TRPV4 activator 4a-phorbol 12,13-didecanoate partly rescued this process and increased survival. In conclusion, TRPV4 plays a critical role in BM adipocyte remodeling induced by leukemia cells, suggesting that targeting TRPV4 may constitute a novel strategy for acute myeloid leukemia therapy.

Meta-analysis of the likelihood of FOXC1 expression in early- and late-stage tumors.

BackgroundAberrations in the expression of the transcription factor forkhead box C1 (FOXC1) have been linked to a number of malignancies. Here, we characterized the relationship between FOXC1 and cancer progression by conducting a meta-analysis of studies that reported the frequency of FOXC1 expression in tumors of different stages (T1, T2, T3, T4).Materials and MethodRelevant articles were retrieved from the Medline database by searching for the terms “FOXC1” and “cancer”; then, the retrieved articles were reviewed individually, and studies that were of multivariate cohort design, evaluated FOXC1 expression via immunohistochemical staining, and assessed the relationship between FOXC1 expression and cancer T-stage were included in our meta-analysis.ResultsOur search terms identified 128 studies, 5 of which met all inclusion criteria. A total of 850 tumor samples were evaluated in the 5 studies; 452 samples were from early-stage (T1-T2) tumors, and 398 were from late-stage (T3-T4) tumors. FOXC1 was expressed in 60.7% (516/850) of all samples, in 54.6% (247/452) of early-stage tumor samples, and in 67.5% (269/398) of late-stage tumor samples. When calculated relative to early-stage samples, the pooled risk for FOXC1 expression in late-stage samples was 1.238 (95% CI = 1.061–1.444, p = 0.007).ConclusionsThe results from our meta-analysis of 5 studies indicate that FOXC1 is 23.8% more likely to be expressed in late-stage tumors than in early-stage tumors.

Forkhead box C1 boosts triple‐negative breast cancer metastasis through activating the transcription of chemokine receptor‐4

The transcription factor forkhead box C1 (FOXC1) has recently been proposed as a crucial regulator of triple‐negative breast cancer (TNBC) and associated with TNBC metastasis. However, the mechanism of FOXC1 in TNBC development and metastasis is elusive. In this study, overexpression of FOXC1 in MDA‐MB‐231 cells significantly enhanced, whereas knockdown of FOXC1 in BT549 cells significantly reduced, the capabilities of TNBC cell invasion and motility in vitro and metastasis to the lung in vivo, when compared to their respective control cells. Mechanistic studies revealed that FOXC1 increased the expression of CXC chemokine receptor‐4 (CXCR4), probably through transcriptional activation. AMD3100, an inhibitor of CXCR4, could block cell migration. In a zebrafish tumor model, AMD3100 could suppress cell invasion and metastasis. In addition, overexpressing CXCR4 in FOXC1‐knockdown BT549 cells increased the capabilities of TNBC cell invasion and motility. In contrast, inhibition of CXCR4 with either AMD3100 or siRNA in MDA‐MB‐231 cells overexpressing FOXC1 reduced the capabilities of invasion and motility. Taken together, our results reveal a potential mechanism for FOXC1‐induced TNBC metastasis.

Meta-analysis of the likelihood of FOXC2 expression in early- and late-stage tumors.

BackgroundAberrations in the expression of the transcription factor forkhead box C2 (FOXC2) have been linked to a number of malignancies. Here, we characterized the relationship between FOXC2 and cancer progression by conducting a meta-analysis of studies that reported the frequency of FOXC2 expression in tumors of different stages (T1, T2, T3, T4).MethodsRelevant articles were retrieved from the Medline database by searching for the terms “FOXC2” and “cancer”; then, the retrieved articles were reviewed individually, and studies that were of multivariate cohort design, evaluated FOXC2 expression via immunohistochemical staining, and assessed the relationship between FOXC2 expression and cancer T-stage were included in our meta-analysis.ResultsOur search terms identified 139 studies, 9 of which met all inclusion criteria. A total of 1433 tumor samples were evaluated in the 9 studies; 596 samples were from early-stage (T1-T2) tumors, and 838 were from late-stage (T3-T4) tumors. FOXC2 was expressed in 46.0% of all samples, in 32.4% of early-stage tumor samples, and in 55.6% of late-stage tumor samples. When calculated relative to early-stage samples, the pooled risk for FOXC2 expression in late-stage samples was 1.367 (95% CI = 1.103–1.695, p = 0.004).ConclusionThe results from our meta-analysis of 9 studies indicate that FOXC2 is 36.7% more likely to be expressed in late-stage tumors than in early-stage tumors.

Role and importance of the expression of transcription factor FOXC2 in cervical cancer.

The aim of the present study was to investigate the relationship between the expression of transcription factor forkhead box C2 (FOXC2) and the clinical features of cervical cancer. A total of 66 patients with cervical cancer, 42 patients with cervical intraepithelial neoplasia (CIN) and 25 patients with cervical inflammation were enrolled. The positive expression rates and expression levels of mRNA of FOXC2, E-cadherin, N-cadherin, vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), Notch protein and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in cervical tissues were detected using immunohistochemistry and RT-PCR. The positive expression rates and expression levels of mRNA of FOXC2, N-cadherin, VEGF, SDF-1, Notch and LYVE-1 in cervical cancer were significantly higher than those in CIN, and those in the inflammatory tissues were the lowest, while the positive expression rate of E-cadherin in cervical cancer was lower than that in CIN, and that in the inflammatory tissues was the highest (P<0.05). The positive expression rates of FOXC2, N-cadherin, VEGF, SDF-1, Notch and LYVE-1 in patients with cervical cancer [human papillomavirus (HPV) positive, squamous cell carcinoma, Stages III–IV, maximal diameter ≥3.8 cm and low differentiation] were increased, and the positive expression rate of E-cadherin was decreased (P<0.05). Correlation analysis revealed that FOXC2 was positively correlated with the positive expression rates of N-cadherin, VEGF, SDF-1, Notch and LYVE-1, and negatively correlated with E-cadherin (P<0.05). In conclusion, the high expression of FOXC2 is correlated with the HPV infection, pathological pattern, clinical stage, tumor diameter and differentiation grade of cervical cancer, which may be involved in the epithelial-mesenchymal transition, vascular and matrix formation, Notch signaling pathway and lymphangiogenesis.

Knockdown of long non-coding RNA XIST increases blood\u2013tumor barrier permeability and inhibits glioma angiogenesis by targeting miR-137

Antiangiogenic therapy plays a significant role in combined glioma treatment. However, poor permeability of the blood–tumor barrier (BTB) limits the transport of chemotherapeutic agents, including antiangiogenic drugs, into tumor tissues. Long non-coding RNAs (lncRNAs) have been implicated in various diseases, especially malignant tumors. The present study found that lncRNA X-inactive-specific transcript (XIST) was upregulated in endothelial cells that were obtained in a BTB model in vitro. XIST knockdown increased BTB permeability and inhibited glioma angiogenesis. The analysis of the mechanism of action revealed that the reduction of XIST inhibited the expression of the transcription factor forkhead box C1 (FOXC1) and zonula occludens 2 (ZO-2) by upregulating miR-137. FOXC1 decreased BTB permeability by increasing the promoter activity and expression of ZO-1 and occludin, and promoted glioma angiogenesis by increasing the promoter activity and expression of chemokine (C–X–C motif) receptor 7b (CXCR7). Overall, the present study demonstrates that XIST plays a pivotal role in BTB permeability and glioma angiogenesis, and the inhibition of XIST may be a potential target for the clinical management of glioma.

FOXC1 Expression in Acute Myeloid Leukemia: Potential Predictor of Disease Relapse and/or Refractory Disease

Background:The mesenchymal transcription factor Forkhead Box C1 (FOXC1), derepressed in hematopoietic stem progenitor cells (HSPCs) in the setting of acute myeloid leukemia (AML), but not in normal HSPCs, was recently demonstrated to play an important role in AML disease onset and progression by blocking myeloid lineage differentiation and enhancing clonogenic potential. Herein we sought to further examine the prognostic value of FOXC1 in AML and whether FOXC1 expression is a potential predictor of disease relapse and/or lack of response to induction chemotherapy in AML.Methods:Expression of FOXC1 mRNA in mixed karyotype AML bone marrow samples was examined using publicly available microarray datasets (n=521, n=244). The clinical significance of FOXC1 gene expression as a prognostic biomarker was evaluated using censored overall survival (OS) and event-free survival (EFS) data. Degree of statistical significance in the univariate and multivariate analyses performed was assessed using log-rank test and Cox regression model, respectively.Results:FOXC1 mRNA expression was a significant predictor of OS on univariate (hazard ratio [HR] 1.592 95% confidence interval [CI] 1.263-2.007, P = 0.0001) and multivariate (HR 1.755 95% CI 1.355-2.273, P < 0.0001) analyses. This effect on OS could be attributed to disease relapse and/or lack of response to induction chemotherapy as FOXC1 mRNA expression also proved to be a significant predictor of EFS on univariate analysis (hazard ratio [HR] 1.539 95% confidence interval [CI] 1.208-1.961, P = 0.0002) and multivariate analysis (HR 1.678 95% CI 1.280-2.201, P = 0.0001), independent of age, FLT3 ITD status, NPM1 status or cytogenetic risk status. Compared to patients who experienced disease remission, FOXC1 expression was significantly elevated in patients experiencing disease relapse following induction chemotherapy (P<0.02), and in patients who were non-responders to induction chemotherapy (P<0.002)Conclusions:FOXC1 expression in AML is an independent prognostic predictor of decreased OS and EFS and is significantly associated with disease relapse and/or refractoriness to induction chemotherapy. A risk of relapse/non-response score that incorporates FOXC1 expression status may prove to be of value in identifying patients at the time of initial diagnosis who are likely to fail induction chemotherapy. Further studies are warranted to explore the prognostic and predictive utility of FOXC1 expression in the clinical management of AML. [Display omitted] DisclosuresRay:Onconostic Technologies, Inc.: Employment. Shah:Onconostic Technologies, Inc.: Employment. Ray:Onconostic Technologies, Inc.: Consultancy, Equity Ownership; 3N Diagnostics Ltd.: Consultancy, Equity Ownership.

FOXC2 augments tumor propagation and metastasis in osteosarcoma.

Osteosarcoma is a highly malignant tumor that contains a small subpopulation of tumor-propagating cells (also known as tumor-initiating cells) characterized by drug resistance and high metastatic potential. The molecular mechanism by which tumor-propagating cells promote tumor growth is poorly understood. Here, we report that the transcription factor forkhead box C2 (FOXC2) is frequently expressed in human osteosarcomas and is important in maintaining osteosarcoma cells in a stem-like state. In osteosarcoma cell lines, we show that anoikis conditions stimulate FOXC2 expression. Downregulation of FOXC2 decreases anchorage-independent growth and invasion in vitro and lung metastasis in vivo, while overexpression of FOXC2 increases tumor propagation in vivo. In osteosarcoma cell lines, we demonstrate that high levels of FOXC2 are associated with and required for the expression of osteosarcoma tumor-propagating cell markers. In FOXC2 knockdown cell lines, we show that CXCR4, a downstream target of FOXC2, can restore osteosarcoma cell invasiveness and metastasis to the lung.

FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential

Adult tissue stem cells (SCs) reside in niches, which orchestrate SC behavior. SCs are typically used sparingly and exist in quiescence unless activated for tissue growth. Whether parsimonious SC use is essential to conserve long-term tissue-regenerating potential during normal homeostasis remains poorly understood. Here, we examine this issue by conditionally ablating a key transcription factor Forkhead box C1 (FOXC1) expressed in hair follicle SCs (HFSCs). FOXC1-deficient HFSCs spend less time in quiescence, leading to markedly shortened resting periods between hair cycles. The enhanced hair cycling accelerates HFSC expenditure, and impacts hair regeneration in aging mice. Interestingly, although FOXC1-deficient HFs can still form a new bulge that houses HFSCs for the next hair cycle, the older bulge is left unanchored. As the new hair emerges, the entire old bulge, including its reserve HFSCs and SC-inhibitory inner cell layer, is lost. We trace this mechanism first, to a marked increase in cell cycle-associated transcripts upon Foxc1 ablation, and second, to a downstream reduction in E-cadherin-mediated inter-SC adhesion. Finally, we show that when the old bulge is lost with each hair cycle, overall levels of SC-inhibitory factors are reduced, further lowering the threshold for HFSC activity. Taken together, our findings suggest that HFSCs have restricted potential in vivo, which they conserve by coupling quiescence to adhesion-mediated niche maintenance, thereby achieving long-term tissue homeostasis.

Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice

Inflammation regulated by interleukin (IL) 8 promotes metastasis of hepatocellular carcinoma (HCC). The transcription factor forkhead box C1 (FOXC1) promotes metastasis by activating the epithelial to mesenchymal transition; its levels in liver tumors have been associated with shorter survival times of patients. We investigated whether FOXC1 activates inflammation signaling pathways in HCC cell lines. We performed studies in the human HCC cell lines Huh-7 and SMMC7721, as well as the metastatic cell lines MHCC97H and HCCLM3. Cell lines were incubated with IL8 and transcription of reporter genes was measured; cells were also incubated with kinase inhibitors. Levels of FOXC1 or IL8 were knocked down with small interfering messenger RNAs in Huh7 cells; cells were analyzed invitro in migration and invasion assays. To study metastasis, HCC cells were injected into flanks of BALB/C nude mice; 4 weeks later, the subcutaneous tumor fragments were collected and implanted into livers of the nude mice, and number and size tumors formed were measured. Chromatin immunoprecipitation assays were used to measure binding of transcription factors promoter regions of genes. We measured levels of FOXC1, IL8, CXCR1, and CCL2 in 2 groups of human HCC tissues collected from the Xijing or Tongji Hospitals in China (n= 690 and n= 312 samples, respectively) using immunohistochemistry. Incubation of HCC cells with IL8 led to increased expression of FOXC1, via activation of phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. Knockdown of FOXC1 in HCC cells that overexpressed IL8 reduced the numbers of metastases formed in mice, compared with cells without FOXC1 knockdown. Transgenic overexpression of FOXC1 in HCC cells with IL8 knockdown increased the numbers of metastases formed in mice compared with cells without FOXC1 overexpression. CXCR1 and CCL2 were direct transcriptional targets of FOXC1. Knockdown of the combination of CXCR1 and CCL2 reduced the invasive activities of HCC cells that overexpress FOXC1 and formation of lung metastases in mice, and transgenic overexpression of CXCR1 increased cell's invasive and metastatic abilities after knockdown of FOXC1. Liver metastases grown from cells that overexpressed FOXC1 were infiltrated by tumor-associated macrophages, and CCL2 knockdown decreased tumor-associated macrophage infiltration; depletion of macrophages from mice significantly reduced growth of metastases by cells that overexpressed FOXC1. In human HCC tissues, level of FOXC1 correlated with levels of IL8 and CXCR1 and CCL2 and infiltration of tumors by macrophage. In multivariate analysis, detection of FOXC1 and CCL2 were independent predictors forpostoperative recurrence of HCC and overall survival. In HCC cell lines, IL8 activates expression of FOXC1 via the phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. FOXC1 expression leads to transactivation of CXCR1 and CCL2, promoting inflammation and the invasive and metastatic abilities of HCC cells.

Lymphangiogenesis in Regenerating Tissue

See related article, pages 1193–1199 The field of lymphangiogenesis has made rapid and exciting developments over the last few years more so than any other field in vascular biology.1 Progress in this field has been long awaited because a number of clinical disorders are associated with impaired lymphatic function. The lymphatic vasculature takes up fluid from the interstitial space, thereby maintaining interstitial fluid balance and providing lymphatic clearance of macromolecules. Abnormal development of lymphatic vessels can give rise to lymphatic malformations, large cystic structures in which lymphatic fluid accumulates. Mutations affecting the vascular endothelial growth factor receptor (VEGFR)-3, or the transcription factor forkhead box C2 (FOXC2), have been implicated in primary congenital lymphedema.2 Insufficient function of lymphatic vessels and lymphedema can also result from chronic inflammation, infection, or trauma. In addition to its function in fluid drainage, the lymphatic system is also an important route for circulating immune cells that function in immune surveillance, but also for the dissemination of metastatic tumor cells in the body.1 Consequently, there are a number of clinical applications for therapeutics that either inhibit or induce lymphangiogenesis. Until recently, the molecular mechanisms of lymphangiogenesis were largely unknown, mainly because molecular markers for lymphatic endothelium were lacking. However, several molecules that are expressed preferentially in lymphatic endothelium have recently been identified, including the VEGFR-3,1 the homeobox gene Prox-1,3 the hyaluronan receptor LYVE-1,4 and the mucin-type transmembrane glycoprotein podoplanin.5 Specific antibodies allowed the isolation and characterization of lymphatic endothelial cells.6 Forced expression of Prox1 in blood vessel endothelial cells caused upregulation of lymphatic markers such as Podoplanin or VEGFR-3, indicating that Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate.6,7 Florence Sabin proposed in 1902 that the lymphatic …