Search

Objective— The lymphatic vasculature is a well-established conduit for metastasis, but the mechanisms by which tumor cells interact with lymphatic endothelial cells (LECs) to facilitate escape remain poorly understood. Elevated levels of the lymphangiogenic peptide adrenomedullin are found in many tumors, and we previously characterized that its expression is necessary for lymphatic vessel growth within both tumors and sentinel lymph nodes and for distant metastasis. Approach and Results— This study used a tumor cell-LEC coculture system to identify a series of adrenomedullin-induced events that facilitated transendothelial migration of the tumor cells through a lymphatic monolayer. High levels of adrenomedullin expression enhanced adhesion of tumor cells to LECs, and further analysis revealed that adrenomedullin promoted gap junction coupling between LECs as evidenced by spread of Lucifer yellow dye. Adrenomedullin also enhanced heterocellular gap junction coupling as demonstrated by Calcein dye transfer from tumor cells into LECs. This connexin-mediated gap junction intercellular communication was necessary for tumor cells to undergo transendothelial migration because pharmacological blockade of this heterocellular communication prevented the ability of tumor cells to transmigrate through the lymphatic monolayer. In addition, treatment of LECs with adrenomedullin caused nuclear translocation of β-catenin, a component of endothelial cell junctions, causing an increase in transcription of the downstream target gene C-MYC . Importantly, blockade of gap junction intercellular communication prevented β-catenin nuclear translocation. Conclusions— Our findings indicate that maintenance of cell–cell communication is necessary to facilitate a cascade of events that lead to tumor cell migration through the lymphatic endothelium.

Levels of the peptide hormone adrenomedullin (AM) are elevated during normal pregnancy, but whether this differs during complications of pregnancy remains unresolved. AM can be quantified by measuring its preprohormone byproduct, midregional pro-adrenomedullin (MR-proADM). MR-pro ADM has shown prognostic value as a biomarker of heart failure, sepsis, and community-acquired pneumonia. Given the relevance of AM to pregnancy, we tested the hypothesis that MR-proADM provides a biomarker for preeclampsia. We find that MR-proADM plasma concentrations are blunted in severe preeclampsia and that MR-proADM is similarly effective as established biomarkers endoglin and placental growth factor at discriminating patients with severe preeclampsia from controls.

SummaryAtypical 7-transmembrane receptors, often called decoy receptors, act promiscuously as molecular sinks to regulate ligand bioavailability and consequently temper the signaling of canonical G protein-coupled receptor (GPCR) pathways. Loss of mammalian CXCR7, the most recently described decoy receptor, results in postnatal lethality due to aberrant cardiac development and myocyte hyperplasia. Here, we provide the molecular underpinning for this proliferative phenotype by demonstrating that the dosage and signaling of adrenomedullin (Adm, gene; AM, protein)—a mitogenic peptide hormone required for normal cardiovascular development—is tightly controlled by CXCR7. To this end, Cxcr7−/− mice exhibit gain-of-function cardiac and lymphatic vascular phenotypes that can be reversed upon genetic depletion of adrenomedullin ligand. In addition to identifying a biological ligand accountable for the phenotypes of Cxcr7−/− mice, these results reveal a previously underappreciated role for decoy receptors as molecular rheostats in controlling the timing and extent of GPCR-mediated cardiac and vascular development.

Adrenomedullin is a highly conserved peptide implicated in a variety of physiological processes ranging from pregnancy and embryonic development to tumor progression. This review highlights past and present studies that have contributed to our current appreciation of the important roles adrenomedullin plays in both normal and disease conditions. We provide a particular emphasis on the functions of adrenomedullin in vascular endothelial cells and how experimental approaches in genetic mouse models have helped to drive the field forward.

Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pH(i)). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochrome c release, and cell death. The chloride channel inhibitor furosemide prevented intracellular alkalinization, Bax translocation, cytochrome c release, and cell death. Translocation of full-length Bid to the mitochondria was also prevented by furosemide. The cleavage product of Bid degradation (truncated Bid, tBid) was not detectable in the mitochondria. Its accumulation in the cytosol was prevented by furosemide. Apoptosis induced by tumor necrosis factor-alpha (TNF) lowered pH(i), an effect also accompanied by Bax translocation, cytochrome c release, and cell killing. Furosemide prevented all of these events. TNF induced a depletion of full-length Bid from the mitochondria and the cytosol but induced an accumulation of mitochondrial tBid. Furosemide only delayed full-length Bid depletion and tBid accumulation. The caspase 8 inhibitor IETD did not prevent the translocation of Bax. Although IETD did inhibit the cleavage of Bid and the accumulation of tBid, cell killing was reduced only slightly. It is concluded that with either staurosporine or TNF a furosemide-sensitive change in pH(i) is linked to Bax translocation, cytochrome c release, and cell killing. With TNF Bax translocation occurs as Bid is depleted and can be dissociated from the accumulation of tBid. With staurosporine a role for full-length Bid in Bax translocation cannot be excluded but is not necessary as evidenced by the data with TNF.

Schlemm’s canal (SC) is a unique vascular structure that functions to maintain fluid homeostasis by draining aqueous humor from the eye into the systemic circulation. The endothelium lining the inner wall of SC has both blood and lymphatic vascular characteristics, thus prompting exploration of the development and regulation of this unique channel. In this issue of the JCI, back-to-back papers by Aspelund et al. and Park et al. detail the mechanisms of SC development, which includes a lymphatic reprogramming that is necessary to maintain proper function. Furthermore, both groups exploit the lymph-like qualities of this canal: they identify VEGF-C as a potential therapeutic for glaucoma and suggest that expression of PROX1, a marker of lymphatic fate, could also serve as a biosensor for monitoring SC integrity. These studies provide substantial insight into the molecular and cellular pathways that govern SC development and reveal that ocular pathology is associated with deregulation of the lymph-like characteristics of SC.

The identification of novel factors that orchestrate the development, growth, and function of the lymphatic vascular system is a relatively new and coveted goal in the broad field of vascular biology. With the increased incidence of lymphedema and the recent recognition of the important roles that lymphatic vessels play in cancer, obesity, and metabolic disorders, there is a strong motivation to identify factors that could serve as pharmacological targets for the therapeutic modulation of lymphatic vessel growth and function.1 Because G protein–coupled receptors constitute the largest proportion of targets for prescribed pharmaceuticals, there is intense interest in revealing novel receptor pathways that could be targeted with highly specific agonists and antagonists.2 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Kim et al3 discover a new player in lymphatic vessels: Apelin signaling—a broadly expressed and multifunctional G protein–coupled receptors pathway that is part of the broader family of adipocytokine signaling.4,5 See accompanying article on page 338 Much of our knowledge on lymphatic vessel growth has come from developmental studies, whereby the establishment of lymphatic progenitor fate, migration, proliferation, and maturation can be elegantly teased apart using genetic approaches in mice and aquatic animal model systems.6,7 Lymphatic progenitors arise from venous endothelial cells, and the process of lymphatic endothelial cell migration away from parental veins can be stereotypically visualized and quantitated in zebrafish. Therefore, identifying key growth factors that are spatiotemporally expressed during this critical point of lymphatic vessel formation provides a strong foundation for the elucidation of new players. This pattern of highly restricted expression is precisely what led Kim et al to explore the possible functions of Apelin signaling in lymphatic development. Previous studies had shown that although the expression of Apelin receptors was broad during early zebrafish development, …

Objectives Plasma levels of adrenomedullin (AM), an anti-inflammatory, vasodilatory peptide hormone, are physiologically elevated during a healthy pregnancy, but it is uncertain whether these levels are altered during complications of pregnancy like preeclampsia. A byproduct of the preprohormone of AM, midregional pro-adrenomedullin (MR-proADM), is produced in a one-to-one ratio with mature AM and can be measured as a surrogate analyte for AM. Therefore, we tested the hypothesis that MR-proADM can serve as a biomarker for preeclampsia. Methods We selected 30 women with preeclampsia and 30 normotensive controls from a prospective study that collected blood and tissue samples as patients presented to Duke University Hospital for delivery. Exclusion criteria were: mild preeclampsia ( n = 5), postpartum plasma collection ( n = 12), twin pregnancies ( n = 3), and smokers ( n = 7). Endoglin, placental growth factor (PlGF), and MR-proADM concentrations were quantified in plasma using a commercial ELISA kit or immunofluorescent assay. Endoglin, PlGF, and MR-proADM were then analyzed as biomarkers of severe preeclampsia by comparing the areas under the curve (AUC) of receiver operating characteristic (ROC) curves generated by logistic regression models of these analytes. Results MR-proADM concentrations were blunted in plasma from women with severe preeclampsia (Matson et al., 2014). In our dataset, endoglin (AUC = 0.73) and MR-proADM (AUC = 0.69) were similarly effective at classifying women with severe preeclampsia and controls, while PlGF was more informative (AUC = 0.85). Endoglin and MR-proADM together (AUC = 0.80) discriminate patients from controls better than they do individually, as do PlGF and MR-proADM together (AUC = 0.87). Conclusions MR-proADM can classify women with severe preeclampsia and controls and may provide a biomarker for severe preeclampsia. However, analysis of MR-proADM levels at earlier gestational ages is needed to determine whether MR-proADM can predict the development of preeclampsia later in pregnancy. Disclosures B. Matson: Research Support Recipient: Ferring Innovation Fellowship. N. Karpinich: None. A. Murtha: None. W. Valdar: None. C.A. Grotegut: None. K.M. Caron: Research Support Recipient; Commercial Interest: Ferring Pharmaceuticals, Inc.

Adrenomedullin (AM) is a potent lymphangiogenic factor that promotes lymphatic endothelial cell (LEC) proliferation through a pharmacologically tractable G-protein-coupled receptor. Numerous types of human cancers have increased levels of AM; however, the functional consequences of this fact have not been characterized. Therefore, we evaluated whether modulating adrenomedullin (Adm) gene dosage within tumor cells affects lymphangiogenesis. Murine Lewis lung carcinoma (LLC) cells that overexpress or underexpress Adm were injected subcutaneously into C57BL/6 mice, and tumors were evaluated for growth and vascularization. A dosage range from ∼10 to 200% of wild-type Adm expression did not affect LLC proliferation in vitro or in vivo, nor did it affect angiogenesis. Notably, the dosage of Adm markedly and significantly influenced tumor lymphangiogenesis. Reduced Adm expression in tumors decreased the proliferation of LECs and the number of lymphatic vessels, while elevated tumor Adm expression led to enlarged lymphatic vessels. Moreover, overexpression of Adm in tumors induced sentinel lymph node lymphangiogenesis and led to an increased incidence of Ki67-positive foci within the lung. These data show that tumor-secreted AM is a critical factor for driving both tumor and lymph node lymphangiogenesis. Thus, pharmacological targeting of AM signaling may provide a new avenue for inhibition of tumor lymphangiogenesis.—Karpinich, N. O., Kechele, D. O., Espenschied, S. T., Willcockson, H. H., Fedoriw, Y., Caron, K. M. Adrenomedullin gene dosage correlates with tumor and lymph node lymphangiogenesis.

Astrocytomas, the most common intracranial malignancies, are a morphologically and molecularly heterogeneous group of brain tumors with potentially dismal patient outcomes for which few effective drugs are available. Genetically engineered mouse (GEM) models of astrocytoma represent a powerful technique for defining the molecular and genetic abnormalities that contribute to tumorigenesis. Based on the genetic aberrations observed in human astrocytomas, we have generated a series of conditional, inducible GEM models of astrocytomas that recapitulate the spectrum of morphological phenotypes of human astrocytomas. However, the extent to which any given GEM model recapitulates the molecular alterations in human tumors must be determined to validate its usefulness in preclinical studies. We are currently pursuing comparative evaluation of primary astrocytomas as formed in GEM and in patients to (1) examine the signaling pathway abnormalities caused by defined genetic lesions in GEM astrocytomas and (2) identify protein biomarkers that can define human astrocytomas that most closely resemble their murine counterparts. To utilize these GEM for combined preclinical evaluation of targeted therapeutic agents and biomarkers predictive of response, we have developed a panel of cell-based assays (CBA) and an orthotopic allograft model of high-grade astrocytomas using primary astrocytes derived from GEM. These tools should prove useful for preclinical drug development studies and provide a link between preclinical drug development in GEM astrocytoma models and rational design of human clinical trials involving only those patients with tumors having similar signaling pathway abnormalities.

Astrocytomas are characterized by diffuse invasion, precluding their complete surgical resection. PTEN, a negative PI3 kinase (PI3K) pathway regulator, is altered in 40-80% of high-grade astrocytomas (HGA), including glioblastoma (GBM). However, its role in astrocytoma invasion remains unclear. Primary astrocytes from six genetically-engineered mouse (GEM) models, with conditional alleles that inactivate Rb (T) and/or Pten (P) and/or constitutively activate Kras (R, KrasG12D) upon Cre recombination, were used to analyze PI3K pathway signaling, proliferation, migration, and invasion in vitro by immunoblot, cell counting, wound healing and time-lapse video microscopy, and collagen invasion, respectively. Gene expression microarrays were used to compare the transcriptomes of GEM astrocytes to human HGA. Tumorigenicity and survival were determined in vivo in orthotopic allograft models. Invasion was assessed by morphometric analysis. Pten ablation increased levels of phospho-Akt and phospho-S6. In cells with both Rb inactivation and Kras activation (TR), complete inactivation of Pten shortened doubling time (DT) by 42% (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4305. doi:1538-7445.AM2012-4305