Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell-specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.
The mammalian lymphatic vasculature is important for returning fluids from the extracellular tissue milieu back to the blood circulation. We showed previously that Prox1 dosage is important for the development of the mammalian lymphatic vasculature. The lack of Prox1 activity results in the complete absence of lymphatic endothelial cells (LECs). In Prox1 heterozygous embryos, the number of LECs is reduced because of a decrease in the progenitor pool in the cardinal vein. This reduction is caused by some progenitor cells being unable to maintain Prox1 expression. In this study, we identified Vegfr3, the cognate receptor of the lymphangiogenic growth factor Vegfc, as a dosage-dependent, direct in vivo target of Prox1. Using various mouse models, we also determined that Vegfr3 regulates Prox1 by establishing a feedback loop necessary to maintain the identity of LEC progenitors and that Vegfc-mediated activation of Vegfr3 signaling is necessary to maintain Prox1 expression in LEC progenitors. We propose that this feedback loop is the main sensing mechanism controlling the number of LEC progenitors and, as a consequence, the number of budding LECs that will form the embryonic lymphatic vasculature.
Resveratrol has beneficial effects on aging, inflammation and metabolism, which are thought to result from activation of the lysine deacetylase, sirtuin 1 (SIRT1), the cAMP pathway, or AMP-activated protein kinase. In this study, we report that resveratrol acts as a pathway-selective estrogen receptor-? (ER?) ligand to modulate the inflammatory response but not cell proliferation. A crystal structure of the ER? ligand-binding domain (LBD) as a complex with resveratrol revealed a unique perturbation of the coactivator-binding surface, consistent with an altered coregulator recruitment profile. Gene expression analyses revealed significant overlap of TNF? genes modulated by resveratrol and estradiol. Furthermore, the ability of resveratrol to suppress interleukin-6 transcription was shown to require ER? and several ER? coregulators, suggesting that ER? functions as a primary conduit for resveratrol activity.DOI: http://dx.doi.org/10.7554/eLife.02057.001.
Lymphatic vasculature is necessary for maintaining fluid homeostasis in vertebrates. During embryogenesis lymphatic endothelial cells originate from the veins as a homogeneous population. These cells undergo a series of changes at the morphological and molecular levels to become mature lymphatic vasculature that consists of lymphatic capillaries, collecting lymphatic vessels and valves. In this article we summarize our current knowledge about these steps and highlight some black boxes that require further clarification.
A 72-year-old man was referred for cataract surgery in the right eye. On slit-lamp biomicroscopy he was noted to have a vertical split in the Descemet's membrane in the left cornea. Based on the appearance, orientation of the split and medical history of forceps delivery, a forceps injury to the left cornea was diagnosed. The break in Descemet's membrane is characteristically vertical due to the horizontal stretching of the globe which occurs with vertical compression of the globe between the orbit and the blade of the obstetric forceps. Forceps injuries are usually unilateral and affect the left eye as the most common fetal head position is left occiput anterior. In the immediate postpartum period the rupture in the Descemet's membrane leads to corneal oedema which eventually disappears leaving the visible edges of the break. This injury also leads to severe left eye astigmatism and secondary amblyopia.
We have explored the isoelectronic replacement of the C?C double bond found at the core of many nonsteroidal estrogen ligands with a simple Schiff base (C?N). Di- and triaryl-substituted imine derivatives were conveniently prepared by the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these imines profiled as ER? agonists but as ER? antagonists, showing preferential reliance on the N-terminal activation function (AF1), which is more active in ER?. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way: by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional coactivators. This work suggests that C?N for C?C substitution might be more widely considered as a general strategy for preparing drug analogues.
Congenital iris defects may usually present either as subtotal aniridia or colobomatous iris defects. Acquired iris defects are secondary to penetrating iris injury, iatrogenic after surgical excision of iris tumours, collateral trauma after anterior segment surgery, or can be postinflammatory in nature. These iris defects can cause severe visual disability in the form of glare, loss of contrast sensitivity, and loss of best corrected visual acuity. The structural loss of iris can be reconstructed with iris suturing, use of prosthetic iris implants, or by a combination of these, depending on the relative amount of residual iris stromal tissue and health of the underlying pigment epithelium. Since the first implant of a black iris diaphragm posterior chamber intraocular lens in 1994, advances in material and design technology over the last decade have led to advances in the prosthetic material, surgical technique, and instrumentation in the field of prosthetic iris implants. In this article, we review the classification of iris defects, types of iris prosthetic devices, implantation techniques, and complications.
To probe the importance of the heterocyclic core of estrogen receptor (ER) ligands, we prepared a series of thiophene-core ligands by Suzuki cross-coupling of aryl boronic acids with bromo-thiophenes and we assessed their receptor binding and cell biological activities. The disposition of the phenol substituents on the thiophene core, at alternate or adjacent sites, and the nature of substituents on these phenols, all contribute to binding affinity and subtype selectivity. Most of the bis(hydroxyphenyl)-thiophenes were ER? selective, whereas the tris(hydroxyphenyl)-thiophenes were ER? selective; analogous furan-core compounds generally have lower affinity and less selectivity. Some diarylthiophenes show distinct superagonist activity in reporter gene assays, giving maximal activities 2-3 times that of estradiol, and modeling suggests that these ligands have a different interaction with a hydrogen-bonding residue in helix-11. Ligand-core modification may be a new strategy for developing ER ligands whose selectivity is based on having transcriptional activity greater than that of estradiol.
A 78-year-old man had uneventful cataract surgery with implantation of a 1-piece plate-haptic intraocular lens in the capsular bag. Seven weeks later, he presented as an emergency with intense fibrinous uveitis and increased intraocular pressure (IOP). Examination revealed an inflammatory capsular block syndrome (CBS) causing fibrinous anterior uveitis and secondary angle-closure glaucoma. The glaucoma resolved and the patients vision improved following neodymium:YAG laser posterior capsulotomy. Inflammatory CBS should be considered in pseudophakic patients presenting with fibrinous anterior uveitis, increased IOP, and secondary angle closure.
Sebaceous gland carcinoma (SGC) arises from the sebaceous glands of the ocular adnexa. It can present as a yellow nodule that may be ulcerated. It can also appear as a plaque-like lesion or as recurrent eyelid inflammation similar to a chalazion. Its ability to spread within the epidermis simulates chronic blepharoconjunctivitis.(1) We present an unusual case of Demodex blepharitis that mimicked eyelid SGC on presentation. A 60-year-old male with generalised eczema was referred to the eye department with chronic bilateral blepharitis worse in the right upper lid. Despite conventional treatment with lid hygiene, oral tetracyclines and topical antibiotic/steroids for several months, the right upper eyelid continued to have lid margin thickening and crusting, meibomian gland dysfunction, telangiectasia, and excoriation (Figures A, B). A course of topical tacrolimus to the peri-orbital skin showed no resolution of symptoms.
Ligand-binding dynamics control allosteric signaling through the estrogen receptor-? (ER?), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ER?-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands showed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways and reveals a new role of the DBD in allosteric control of ER?-mediated signaling.
Arteries, veins, and lymphatic vessels are functionally linked, and their physical interaction is tightly regulated. The lymphatic vessels communicate with the blood vessels only at the junction of the jugular and subclavian veins. Here, we characterize the embryonic lymphovenous valves controlling this vital communication and show that they are formed by the intercalation of lymphatic endothelial cells (LECs) with a subpopulation of venous endothelial cells (ECs) at the junction of the jugular and subclavian veins. We found that unlike LEC progenitors, which move out from the veins and differentiate into mature LECs, these Prox1-expressing ECs remain in the veins and do not acquire LEC features. We demonstrate that the development of this Prox1-expressing venous EC population, and therefore of lymphovenous valves, requires two functional copies of Prox1, as the valves are absent in Prox1 heterozygous mice. We show that this is due to a defect in the maintenance of Prox1 expression in venous ECs and LEC progenitors promoted by a reduction in Coup-TFII/Prox1 complex formation. This is the first report describing the molecular mechanism controlling lymphovenous communication.
A 7-year-old Caucasian girl presented with a pigmented lesion on the left bulbar conjunctiva that increased in size from 1?mm to 4?mm over a 12-month period. She underwent excision biopsy and reconstruction of the ocular surface with amniotic membrane graft. Histopathology showed the naevus was composed of somewhat swollen naevus cells with clear cytoplasm and central nucleus. These vacuolated naevus cells were approximately 40?µm in diameter. Over 90% of the cells in the naevus were composed of these swollen cells. Immunohistochemical staining was positive for S100 and Melan-A. This case illustrates that balloon cells may be observed in conjunctival naevi at a previously unreported pre-pubescent age. Awareness of ballon cell naevus is important to avoid clinical and histological pitfalls in diagnosis.
Conjunctival lymphangiectasia is an uncommon clinical condition in which there is dilatation of lymphatic channels in the bulbar conjunctiva. Conjunctival lymphangiectasia is a rarely appreciated ocular surface disorder that typically occurs as a secondary phenomenon in response to local lymphatic scarring or distal obstruction. Conjunctival lymphangiectasia can either be unilateral or bilateral with focal or diffuse bulbar chemosis. We present 11 cases of biopsy-proven conjunctival lymphangiectasia. Of the 11 cases, 3 presented with bilateral diffuse bulbar chemosis, 1 had diffuse unilateral chemosis, and the remaining 7 presented with focal (<90°) bulbar chemosis. Three of these cases had co-existing pterygium, and one case presented with focal bulbar chemosis and a conjunctival keratin horn. All underwent surgical excision of the involved conjunctiva, either with no graft (n = 6), combined with amniotic membrane transplant (n = 3), or combined with conjunctival autograft (n = 2).
Lymph node metastasis constitutes a key event in tumor progression. The molecular control of this process is poorly understood. Heparan sulfate is a linear polysaccharide consisting of unique sulfate-modified disaccharide repeats that allow the glycan to bind a variety of proteins, including chemokines. While some chemokines may drive lymphatic trafficking of tumor cells, the functional and genetic importance of heparan sulfate as a possible mediator of chemokine actions in lymphatic metastasis has not been reported.
The homeobox gene Prox1 is crucial for mammalian lymphatic vascular development. In the absence of Prox1, lymphatic endothelial cells (LECs) are not specified. The maintenance of LEC identity also requires the constant expression of Prox1. However, the mechanisms controlling the expression of this gene in LECs remain poorly understood. The SRY-related gene Sox18 is required to induce Prox1 expression in venous LEC progenitors. Although Sox18 is also expressed in embryonic arteries, these vessels do not express Prox1, nor do they give rise to LECs. This finding suggests that some venous endothelial cell-specific factor is required for the activation of Prox1. Here we demonstrate that the nuclear hormone receptor Coup-TFII is necessary for the activation of Prox1 in embryonic veins by directly binding a conserved DNA domain in the regulatory region of Prox1. In addition, we show that the direct interaction between nuclear hormone receptors and Prox1 is also necessary for the maintenance of Prox1 expression during early stages of LEC specification and differentiation.
This study elucidates the role of E6-associated protein, E6-AP (a dual function steroid hormone receptor coactivator and ubiquitin-protein ligase) in the regulation of PI3K-Akt signaling pathway, prostate gland growth and proliferation. Here, we report the generation of transgenic mice and prostate cancer cell line, LNCaP cells that overexpress E6-AP protein. Using these models we show that the levels of total Akt and phosphorylated Akt (active Akt) are increased in E6-AP overexpressing prostate gland and LNCaP cells suggesting that E6-AP regulates the PI3K-Akt signaling pathway. The prostate glands in our transgenic mice are ~20% larger and produce preneoplastic lesions at the age of 18 months. Our data also suggest that E6-AP modulates PI3K-Akt signaling pathway by both androgen-independent and -dependent mechanisms. In the androgen-independent mechanism, E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. Further, we show that E6-AP, a known coactivator of AR, amplifies the androgen-dependent activation of PI3K-Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased cell size and proliferation. Overall our data suggests that E6-AP regulates both the positive and negative modulators of the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and decreased apoptosis.This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!
Lineage commitment and differentiation into mature cell types are mostly considered to be unidirectional and irreversible processes. However, recent results have challenged this by showing that terminally differentiated cell types can be reprogrammed into other cell types, an important step towards devising strategies for gene therapy and tissue regeneration. In this Review, we summarize recent data on the earliest steps in the development of the mammalian lymphatic vasculature: the specification of lymphatic endothelial cells (LECs). We elaborate on a developmental model that integrates the different steps leading to LEC differentiation and lymphatic network formation, discuss evidence that suggests that LEC fate is plastic, and consider the potentially far-reaching implications of the ability to convert one cell type into another.
The femtosecond laser is gaining popularity as a safe and effective alternative to the microkeratome for creating corneal flaps during LASIK. Rarely, gas bubble formation in the anterior chamber during laser flap creation can interfere with the eye tracking and iris registration software during the subsequent excimer laser ablation. Surgical steps to ensure successful eye tracking and iris registration in such cases are described in this report.
A 67-year-old man with a history of multiple myeloma (treated with chemotherapy) was referred with a left hyperaemic conjunctival lesion covering almost 360° of the limbus and extending onto the corneal surface. Conjunctival biopsy revealed conjunctival intraepithelial neoplasia. Initial treatment consisted of topical and intralesional injections of interferon ?-2b. The patient subsequently developed limbal stem cell deficiency resulting in a persistent non-healing corneal epithelial defect. This was successfully managed with total excisional biopsy of the lesion, combined with limbal stem cell autograft (from the fellow eye) and amniotic membrane transplantation. Histopathology revealed a conjunctival squamous cell carcinoma. The corneal epithelium completely healed postoperatively and there is no evidence of tumour recurrence at 1 year follow-up. This case highlights a rare case of advanced ocular surface neoplasia causing secondary limbal stem cell deficiency. Medical and surgical management of ocular surface neoplasia with limbal stem cell transplantation is effective in treating such cases.
Safety pharmacological studies need to be performed according to ICH S7A Guidelines for finished formulations that substantially alter the pharmacokinetics and/or pharmacodynamics of the active substance in comparison to formulations previously tested (i. e. through active excipients such as penetration enhancers, liposomes, and other changes such as polymorphous system). In the present study, amorphous formulation of celecoxib (CAS 169590-42-5), a new patented formulation with altered pharmacokinetic profile was investigated in comparison with the standard crystalline celecoxib (CEL) for its undesirable pharmacodynamic effects using certain safety pharmacological studies. The effects of the new formulation on vital functions using safety pharmacology core battery like central nervous system (CNS) (functional observation battery, locomotor activity, and motor coordination) and cardiovascular system (CVS) (blood pressure, heart rate and QT interval) were investigated in laboratory rodents. In addition, supplementary safety pharmacology study on gastrointestinal system (GIT) (gastric injury potential, gastric secretion) was also carried out. Oral administration of a single dose of the amorphous celecoxib formulation (CF) varying of 50, 150 and 500 mg/kg was studied in comparison with vehicle treated control and crystalline celecoxib in animals. The maximum tolerated dose (MTD) was identified by administration of insufferable doses of amorphous formulation, extended up to 2000 mg/kg during the experiments on physiological parameters. There were no CNS and GIT safety concerns raised with respect to use of CF except the arrythmogenic risk associated with QT interval prolongation upon the high dose of CF.
Compounds that block estrogen action through the estrogen receptor (ER) or downregulate ER levels are useful for the treatment of breast cancer and endocrine disorders. In our search for structurally novel estrogens having three-dimensional core scaffolds, we found some compounds with a 7-oxabicyclo[2.2.1]heptene core that bound well to the ERs. The best of these compounds, a phenyl sulfonate ester (termed OBHS for oxabicycloheptene sulfonate), was a partial antagonist on both ER? and ER?. Although OBHS bears no structural resemblance to other estrogen antagonists, it appears to achieve its partial antagonist character by stabilizing a novel conformation of the ER that involves a significant distortion of helix-11. To enhance the antagonist properties of these oxabicyclo[2.2.1]heptane core ligands, we expanded the functional diversity of OBHS by replacing the sulfonate with secondary or tertiary sulfonamides (-SO(2)NR-), isoelectronic and potentially isostructural molecular replacements. An array of 16 OBHS sulfonamide analogues were prepared through a Diels-Alder reaction of a 3,4-diarylfuran using various N-aryl vinyl sulfonamide dienophiles. While the more polar secondary sulphonamides were weak ligands, certain of the tertiary sulfonamides had very good ER binding affinity. In HepG2 cell reporter gene assays, the sulphonamides had moderate potency, but they showed lower intrinsic transcriptional activity on ER? than the selective estrogen receptor modulator (SERM) hydroxytamoxifen or OBHS, and they were inverse agonists on ER?. Thus, the behaviour of these OBH-sulfonamides more closely mirrors the activity of full antagonists like the drug fulvestrant (ICI 182?780), and their greater antagonist biocharacter appears to arise from an accentuated distortion of helix-11.
The lymphatic vasculature preserves tissue fluid balance by absorbing fluid and macromolecules and transporting them to the blood vessels for circulation. The stepwise process leading to the formation of the mammalian lymphatic vasculature starts by the expression of the gene Prox1 in a subpopulation of blood endothelial cells (BECs) on the cardinal vein (CV) at approximately E9.5. These Prox1-expressing lymphatic endothelial cells (LECs) will exit the CV to form lymph sacs, primitive structures from which the entire lymphatic network is derived. Until now, no conclusive information was available regarding the cellular processes by which these LEC progenitors exit the CV without compromising the veins integrity. We determined that LECs leave the CV by an active budding mechanism. During this process, LEC progenitors are interconnected by VE-cadherin-expressing junctions. Surprisingly, we also found that Prox1-expressing LEC progenitors were present not only in the CV but also in the intersomitic vessels (ISVs). Furthermore, as LEC progenitors bud from the CV and ISVs into the surrounding mesenchyme, they begin expressing the lymphatic marker podoplanin, migrate away from the CV, and form the lymph sacs. Analyzing this process in Prox1-null embryos revealed that Prox1 activity is necessary for LEC progenitors to exit the CV.
Human vascular malformations cause disease as a result of changes in blood flow and vascular hemodynamic forces. Although the genetic mutations that underlie the formation of many human vascular malformations are known, the extent to which abnormal blood flow can subsequently influence the vascular genetic program and natural history is not. Loss of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) resulted in a vascular malformation that directed blood flow through mesenteric lymphatic vessels after birth in mice. Mesenteric vessels in the position of the congenital lymphatic in mature Slp76-null mice lacked lymphatic identity and expressed a marker of blood vessel identity. Genetic lineage tracing demonstrated that this change in vessel identity was the result of lymphatic endothelial cell reprogramming rather than replacement by blood endothelial cells. Exposure of lymphatic vessels to blood in the absence of significant flow did not alter vessel identity in vivo, but lymphatic endothelial cells exposed to similar levels of shear stress ex vivo rapidly lost expression of PROX1, a lymphatic fate-specifying transcription factor. These findings reveal that blood flow can convert lymphatic vessels to blood vessels, demonstrating that hemodynamic forces may reprogram endothelial and vessel identity in cardiovascular diseases associated with abnormal flow.
Anterior lamellar keratoplasty (ALK) is indicated in patients with anterior corneal opacities. Benefits over penetrating keratoplasty include quicker visual rehabilitation, less postoperative astigmatism, and preservation of the host endothelium, thus minimizing the chances of graft rejection. A rare complication of lamellar corneal surgery is infectious interface keratitis between the donor and host tissue. We report a case of infectious interface keratitis following automated ALK successfully treated medically and by removal of the ALK disk, eventually having a deep anterior lamellar keratoplasty with good visual recovery.
Endothelial cells of initial lymphatics have discontinuous button-like junctions (buttons), unlike continuous zipper-like junctions (zippers) of collecting lymphatics and blood vessels. Buttons are thought to act as primary valves for fluid and cell entry into lymphatics. To learn when and how buttons form during development and whether they change in disease, we examined the appearance of buttons in mouse embryos and their plasticity in sustained inflammation. We found that endothelial cells of lymph sacs at embryonic day (E)12.5 and tracheal lymphatics at E16.5 were joined by zippers, not buttons. However, zippers in initial lymphatics decreased rapidly just before birth, as buttons appeared. The proportion of buttons increased from only 6% at E17.5 and 12% at E18.5 to 35% at birth, 50% at postnatal day (P)7, 90% at P28, and 100% at P70. In inflammation, zippers replaced buttons in airway lymphatics at 14 and 28 days after Mycoplasma pulmonis infection of the respiratory tract. The change in lymphatic junctions was reversed by dexamethasone but not by inhibition of vascular endothelial growth factor receptor-3 signaling by antibody mF4-31C1. Dexamethasone also promoted button formation during early postnatal development through a direct effect involving glucocorticoid receptor phosphorylation in lymphatic endothelial cells. These findings demonstrate the plasticity of intercellular junctions in lymphatics during development and inflammation and show that button formation can be promoted by glucocorticoid receptor signaling in lymphatic endothelial cells.
Previously, we discovered estrogen receptor (ER) ligands with a novel three-dimensional oxabicyclo[2.2.1]heptene core scaffold and good ER binding affinity act as partial agonists via small alkyl ester substitutions on the bicyclic core that indirectly modulate the critical switch helix in the ER ligand binding domain, helix 12, by interactions with helix 11. This contrasts with the mechanism of action of tamoxifen, which directly pushes helix 12 out of the conformation required for gene activation. We now report that a much larger substitution can be tolerated at this position of the bicyclic core scaffold, namely a phenyl sulfonate group, which defines a novel binding epitope for the estrogen receptor. We prepared an array of 14 oxabicycloheptene sulfonates, varying the phenyl sulfonate group. As with the parent compound, 5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester (OBHS), these compounds showed preferential affinity for ER?, and the disposition and size of the phenyl substituents were important determinants of the binding affinity and selectivity of these compounds, with those having ortho substituents giving the highest, and para substituents the lowest affinities for ER?. A few analogues exhibit ER? binding affinities that are comparable to or, in the case of the ortho-chloro analogue, higher than that of OBHS itself. In cell-based studies, we found several compounds with activity profiles comparable to tamoxifen, but acting entirely as indirect antagonists, allosterically interfering with recruitment of coactivator proteins to the receptor. Thus, the OBHS binding epitope represents a novel approach to the development of estrogen receptor antagonists via an indirect mechanism of antagonism.
Notch signaling is active in mouse cochlear prosensory progenitors but declines in differentiating sensory hair cells (HCs). Overactivation of the Notch1 intracellular domain (NICD) in progenitors blocks HC fate commitment and/or differentiation. However, it is not known whether reactivation of NICD in differentiating HCs also interrupts their developmental program and reactivates its downstream targets.
RASA1 (also known as p120 RasGAP) is a Ras GTPase-activating protein that functions as a regulator of blood vessel growth in adult mice and humans. In humans, RASA1 mutations cause capillary malformation-arteriovenous malformation (CM-AVM); whether it also functions as a regulator of the lymphatic vasculature is unknown. We investigated this issue using mice in which Rasa1 could be inducibly deleted by administration of tamoxifen. Systemic loss of RASA1 resulted in a lymphatic vessel disorder characterized by extensive lymphatic vessel hyperplasia and leakage and early lethality caused by chylothorax (lymphatic fluid accumulation in the pleural cavity). Lymphatic vessel hyperplasia was a consequence of increased proliferation of lymphatic endothelial cells (LECs) and was also observed in mice in which induced deletion of Rasa1 was restricted to LECs. RASA1-deficient LECs showed evidence of constitutive activation of Ras in situ. Furthermore, in isolated RASA1-deficient LECs, activation of the Ras signaling pathway was prolonged and cellular proliferation was enhanced after ligand binding to different growth factor receptors, including VEGFR-3. Blockade of VEGFR-3 was sufficient to inhibit the development of lymphatic vessel hyperplasia after loss of RASA1 in vivo. These findings reveal a role for RASA1 as a physiological negative regulator of LEC growth that maintains the lymphatic vasculature in a quiescent functional state through its ability to inhibit Ras signal transduction initiated through LEC-expressed growth factor receptors such as VEGFR-3.
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