Unique functions for Notch4 in murine embryonic lymphangiogenesis.

In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

Lymphatic Endothelial Cell Defects in Congenital Cardiac Patients With Postoperative Chylothorax.

OBJECTIVES: Chylothorax following cardiac surgery for congenital cardiac anomalies is a complication associated with severe morbidities and mortality. We hypothesize that there are intrinsic defects in the lymphatics of congenital cardiac patients. METHODS: Postsurgical chylothorax lymphatic endothelial cells (pcLECs) (n = 10) were isolated from the chylous fluid from congenital cardiac defect patients, and characterized by fluorescent-activated cell sorting, immunofluorescent staining, and quantitative RT-PCR. Results were compared to normal human dermal lymphatic endothelial cells (HdLECs). pcLECs (n = 3) and HdLECs were xenografted into immunocompromised mice. Implants and postoperative chylothorax patient's pulmonary tissues were characterized by immunostaining for lymphatic endothelial proteins. RESULTS: pcLECs expressed endothelial markers VECADHERIN, CD31, VEGFR2, lymphatic endothelial markers PROX1, podoplanin, VEGFR3, and progenitor endothelial markers CD90 and CD146. However, pcLECs had key differences relative to HdLECs, including altered expression and mislocalization of junctional proteins (VECADHERIN and CD31), and essential endothelial proteins, VEGFR2, VEGFR3, and PROX1. When xenografted in mice, pcLECs formed dilated lymphatic channels with poor cell-cell association. Similar to congenital lymphatic anomalies, the pulmonary lymphatics were dilated in a patient who developed postoperative chylothorax after cardiac surgery. CONCLUSIONS: Recent studies have shown that some postoperative chylothoraces in congenital cardiac anomalies are associated with anatomical lymphatic defects. We found that pcLECs have defects in expression and localization of proteins necessary to maintain lymphatic specification and function. This pcLEC phenotype is similar to that observed in lymphatic endothelial cells from congenital lymphatic anomalies. Co-existence of lymphatic anomalies should be considered as a feature of congenital cardiac anomalies.

Nitric Oxide Reverses the Position of the Heart during Embryonic Development.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays crucial roles in cardiac homeostasis. Adult cardiomyocyte specific overexpression of eNOS confers protection against myocardial-reperfusion injury. However, the global effects of NO overexpression in developing cardiovascular system is still unclear. We hypothesized that nitric oxide overexpression affects the early migration of cardiac progenitor cells, vasculogenesis and function in a chick embryo. Vehicle or nitric oxide donor DEAN (500 mM) were loaded exogenously through a small window on the broad side of freshly laid egg and embryonic development tracked by live video-microscopy. At Hamburg Hamilton (HH) stage 8, the cardiac progenitor cells (CPC) were isolated and cell migration analysed by Boyden Chamber. The vascular bed structure and heart beats were compared between vehicle and DEAN treated embryos. Finally, expression of developmental markers such as BMP4, Shh, Pitx2, Noggin were measured using reverse transcriptase PCR and in-situ hybridization. The results unexpectedly showed that exogenous addition of pharmacological NO between HH stage 7⁻8 resulted in embryos with situs inversus in 28 out of 100 embryos tested. Embryos treated with NO inhibitor cPTIO did not have situs inversus, however 10 embryos treated with L-arginine showed a situs inversus phenotype. N-acetyl cysteine addition in the presence of NO failed to rescue situs inversus phenotype. The heart beat is normal (120 beats/min) although the vascular bed pattern is altered. Migration of CPCs in DEAN treated embryos is reduced by 60% compared to vehicle. BMP4 protein expression increases on the left side of the embryo compared to vehicle control. The data suggests that the NO levels in the yolk are important in turning of the heart during embryonic development. High levels of NO may lead to situs inversus condition in avian embryo by impairing cardiac progenitor cell migration through the NO-BMP4-cGMP axis.

Myeloid Wnt ligands are required for normal development of dermal lymphatic vasculature.

Resident tissue myeloid cells play a role in many aspects of physiology including development of the vascular systems. In the blood vasculature, myeloid cells use VEGFC to promote angiogenesis and can use Wnt ligands to control vascular branching and to promote vascular regression. Here we show that myeloid cells also regulate development of the dermal lymphatic vasculature using Wnt ligands. Using myeloid-specific deletion of the WNT transporter Wntless we show that myeloid Wnt ligands are active at two distinct stages of development of the dermal lymphatics. As lymphatic progenitors are emigrating from the cardinal vein and intersomitic vessels, myeloid Wnt ligands regulate both their numbers and migration distance. Later in lymphatic development, myeloid Wnt ligands regulate proliferation of lymphatic endothelial cells (LEC) and thus control lymphatic vessel caliber. Myeloid-specific deletion of WNT co-receptor Lrp5 or Wnt5a gain-of-function also produce elevated caliber in dermal lymphatic capillaries. These data thus suggest that myeloid cells produce Wnt ligands to regulate lymphatic development and use Wnt pathway co-receptors to regulate the balance of Wnt ligand activity during the macrophage-LEC interaction.

A comparative study of NONOate based NO donors: spermine NONOate is the best suited NO donor for angiogenesis.

Nitric oxide (NO) is a known modulator of angiogenesis. The NONOate subfamily of NO donors has long been used in experimental and clinical studies to promote angiogenesis. However, no studies have been conducted yet to compare the angiogenesis potential of these NO donors in respect to their pattern of NO release. We hypothesize that having different pattern of NO release, each of the NO donors in NONOate subfamily can promote key stages of angiogenesis in differential manner. To verify our hypothesis, NO donors with half life ranging from seconds to several hours and having very different pattern of NO release were selected to evaluate their efficacy in modulating angiogenesis. Endothelial tube formation using EAhy926 cells was maximally increased by Spermine NONOate (SP) treatment. SP treatment maximally induced both ex vivo and in vivo angiogenesis using egg yolk and cotton plug angiogenesis models respectively. Experiment using chick embryo partial ischemia model revealed SP as the best suited NO donor to recover ischemia driven hampered angiogenesis. The present study elaborated that differential release pattern of NO by different NO donors can modulate angiogenesis differentially and also suggested that SP have a unique pattern of NO release that best fits for angiogenesis.

NO (nitric oxide): the ring master.

The migration and proliferation of endothelial cells affect the process of angiogenesis or the formation of blood vessels. Endothelial cells interact with each other to form ring-like structures in monolayers and tubular structures in matrigels. However, the transit phase between the individual endothelial cells and fully formed tubular structures is yet to be established. Guided by imaging, Western blot analysis, drug perturbation studies and siRNA studies we validate that endothelial ring structures are the fundamental and monomeric units of capillary tubes and nitric oxide is implicated in their fabrication. Giving input from experimental data, we used bagging classifier and information-gain to determine some of the physical and chemical parameters that define these biological structures. Further, we elucidated the implications of endothelial nitric oxide synthase and the NO/sGC/cGMP pathway in the formation of endothelial rings. We conclude that, formation of endothelial ring structure is important for angiogenesis and is mediated by the NO/sGC/cGMP pathway; and further endothelial rings can be used as in vitro models to study angiogenesis.

Simulated microgravity promotes nitric oxide-supported angiogenesis via the iNOS-cGMP-PKG pathway in macrovascular endothelial cells.

Angiogenesis is a physiological process involving the growth of blood vessel in response to specific stimuli. The present study shows that limited microgravity treatments induce angiogenesis by activating macrovascular endothelial cells. Inhibition of nitric oxide production using pharmacological inhibitors and inducible nitric oxide synthase (iNOS) small interfering ribo nucleic acid (siRNA) abrogated microgravity induced nitric oxide production in macrovascular cells. The study further delineates that iNOS acts as a molecular switch for the heterogeneous effects of microgravity on macrovascular, endocardial and microvascular endothelial cells. Further dissection of nitric oxide downstream signaling confirms that simulated microgravity induces angiogenesis via the cyclic guanosine monophosphate (cGMP)-PKG dependent pathway.

Inhibition of dynamin-2 confers endothelial barrier dysfunctions by attenuating nitric oxide production.

Hypoxia induces barrier dysfunctions in endothelial cells. Nitric oxide is an autacoid signalling molecule that confers protection against hypoxia-mediated barrier dysfunctions. Dyn-2 (dynamin-2), a large GTPase and a positive modulator of eNOS (endothelial nitric oxide synthase), plays an important role in maintaining vascular homeostasis. The present study aims to elucidate the role of dyn-2 in hypoxia-mediated leakiness of the endothelial monolayer in relation to redox milieu. Inhibition of dyn-2 by transfecting the cells with K44A, a dominant negative construct of dyn-2, elevated leakiness of the endothelial monolayer under hypoxia. Sodium nitroprusside (nitric oxide donor) and uric acid (peroxynitrite quencher) were used to evaluate the role of nitric oxide and peroxynitrite in maintaining endothelial barrier functions under hypoxia. Administration of nitric oxide and uric acid recovered hypoxia-mediated leakiness of K44A-overexpressed endothelial monolayer. Our study confirms that inhibition of dyn-2 induces leakiness in the endothelial monolayer by increasing the load of peroxynitrite under hypoxia.

Shear stress promotes nitric oxide production in endothelial cells by sub-cellular delocalization of eNOS: A basis for shear stress mediated angiogenesis.

This study aims to investigate the role of shear stress in cellular remodeling and angiogenesis with relation to nitric oxide (NO). We observed a 2-fold increase in endothelial cell (EC) migration in relation to actin re-arrangements under 15 dyne/cm(2) shear stress. Blocking NO production inhibited the migration and ring formation of ECs by 6-fold and 5-fold, respectively under shear stress. eNOS-siRNA knockdown technique also ascertained a 3-fold reduction in shear stress mediated ring formation. In ovo artery ligation model with a half and complete flow block for 30 min showed a reduction of angiogenesis by 50% and 70%, respectively. External stimulation with NO donor showed a 2-fold recovery in angiogenesis under both half and complete flow block conditions. NO intensity clustering studies by using Diaminofluorescein diacetate (DAF-2DA) probed endothelial monolayer depicted pattern-changes in NO distribution and cluster formation of ECs under shear stress. Immunofluorescence and live cell studies revealed an altered sub-cellular localization pattern of eNOS and phospho-eNOS under shear stress. In conclusion, shear-induced angiogenesis is mediated by nitric oxide dependent EC migration.

Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells.

Microgravity causes endothelium dysfunctions and vascular endothelium remodeling in astronauts returning from space flight. Cardiovascular deconditioning occurs as a consequence of an adaptive response to microgravity partially due to the effects exerted at cellular level. Directional migration of endothelial cell which are central in maintaining the structural integrity of vascular walls is regulated by chemotactic, haptotactic, and mechanotactic stimuli which are essential for vasculogenesis. We explored the migration property of transformed endothelial cells (EC) exposed to 2-h microgravity, simulated using a three-dimensional clinostat constructed based on blueprint published by the Fokker Space, Netherlands. Migration of EC was measured using the scrap wound healing in the presence or absence of actin polymerization inhibitor-cytochalasin D (CD) in Eahy926 cell lines. Simulated microgravity increased cellular migration by 25% while CD-blocked microgravity induced cellular migration. The key migratory structures of cells, filopodia and lamellipodia, formed by EC were more in simulated microgravity compared to gravity. Parallel experiments with phalloidin and diaminorhodamine-4M (DAR-4M) showed that simulated microgravity caused actin rearrangements that lead to 25% increase in nitric oxide production. Further nitric oxide measurements showed a higher nitric oxide production which was not abrogated by phosphoinositol 3 kinase inhibitor (Wortmanin). Bradykinin, an inducer of nitric oxide, prompted two folds higher nitric oxide production along with simulated microgravity in a synergistic manner. We suggest that limited exposure to simulated microgravity increases Eahy926 cell migration by modulating actin and releasing nitric oxide.

Secreted frizzled-related protein 4: an angiogenesis inhibitor.

Wnt signaling is involved in developmental processes, cell proliferation, and cell migration. Secreted frizzled-related protein 4 (sFRP4) has been demonstrated to be a Wnt antagonist; however, its effects on endothelial cell migration and angiogenesis have not yet been reported. Using various in vitro assays, we show that sFRP4 inhibits endothelial cell migration and the development of sprouts and pseudopodia as well as disrupts the stability of endothelial rings in addition to inhibiting proliferation. sFRP4 interfered with endothelial cell functions by antagonizing the canonical Wnt/beta-catenin signaling pathway and the Wnt/planar cell polarity pathway. Furthermore, sFRP4 blocked the effect of vascular endothelial growth factor on endothelial cells. sFRP4 also selectively induced apoptotic events in endothelial cells by increasing cellular levels of reactive oxygen species. In vivo assays demonstrated a reduction in vascularity after sFRP4 treatment. Most importantly, sFRP4 restricted tumor growth in mice by interfering with endothelial cell function. The data demonstrate sFRP4 to be a potent angiogenesis inhibitor that warrants further investigation as a therapeutic agent in the control of angiogenesis-associated pathology.

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase.

BACKGROUND AND PURPOSE: Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets. EXPERIMENTAL APPROACH: Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC). KEY RESULTS: Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis. CONCLUSION AND IMPLICATIONS: Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.

Cadmium attenuates bradykinin-driven nitric oxide production by interplaying with the localization pattern of endothelial nitric oxide synthase.

Cadmium, a ubiquitous heavy metal, interferes with endothelial functions and angiogenesis. Bradykinin is a Ca-mobilizing soluble peptide that acts via nitric oxide to promote vasodilation and capillary permeability. The objective of the present study was to explore the Cd implications in bradykinin-dependent endothelial functions. An egg yolk angiogenesis model was employed to evaluate the effect of Cd on bradykinin-induced angiogenesis. The results demonstrate that 100 nmol/L Cd attenuated bradykinin-dependent angiogenesis. The results of the in vitro wound healing and tube formation assays by using EAhy 926, a transformed endothelial cell line, suggest that Cd blocked bradykinin-mediated endothelial migration and tube formation by 38% and 67%, respectively, while nitric oxide supplementation could reverse the effect of Cd on bradykinin-induced endothelial migration by 94%. The detection of nitric oxide by using a DAF-2DA fluorescent probe, Griess assay, and ultrasensitive electrode suggests that Cd blocked bradykinin-induced nitric oxide production. Fluorescence imaging of eNOS-GFP transfected endothelial cells, immunofluorescence, and Western blot studies of Cd and bradykinin-treated cells show that Cd interfered with the localization pattern of eNOS, which possibly attenuates nitric oxide production in part. Additionally, Ca imaging of Cd- and bradykinin-treated cells suggests that Cd blocked bradykinin-dependent Ca influx into the cells, thus partially blocking Ca-dependent nitric oxide production in endothelial cells. The results of this study conclude that Cd blunted the effect of bradykinin by interfering with the Ca-associated NOS activity specifically by impeding subcellular trafficking of eNOS.

Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase.

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.

Activated pericyte attenuates endothelial functions: nitric oxide-cGMP rescues activated pericyte-associated endothelial dysfunctions.

Hepatic stellate cells are liver-specific pericytes and exist in close proximity with endothelial cells. The activation of liver pericytes is intrinsic to liver pathogenesis, and leads to endothelial dysfunction, including the low bioavailability of nitric oxide (NO). However, the role of nitric oxide in pericyte-endothelium cross-talk has not yet been elucidated. This work examines the cellular mechanism of action of NO in pericyte-mediated endothelial dysfunction. We used in vitro coculture and conditioned medium systems to study the effects of activated liver pericytes on endothelial function, and an egg yolk vascular bed model was used to study the effects of activated pericytes on angiogenesis. This study also demonstrates that activated pericytes attenuate the migration, proliferation, permeability, and NO production of endothelial cells. Our results demonstrate that activated pericytes restrict angiogenesis in egg yolk vascular bed models, and NO supplementation recovers 70% of the inhibition. Our results also demonstrate that supplementation with NO, sildenafil citrate (phosphodiesterase inhibitor), and 8-bromo-cGMP (cGMP analog) partially recovers activated-pericyte-mediated endothelium dysfunction. We conclude that NO-cGMP alleviates activated-pericyte-associated endothelial dysfunction, including angiogenesis, in a cGMP-dependent manner.