KDR Expression Research Articles

Molecular mechanism of inhibition of the abnormal proliferation of human umbilical vein endothelial cells by hydroxysafflor-yellow A

Context It has been found that hydroxysafflor-yellow A (HSYA) inhibits angiogenesis and the proliferation of abnormal human umbilical vein endothelial cells (HUVECs) in our previous study; however, the mechanism is still unclear.Objective This study investigates the mechanisms of HSYA inhibiting abnormal proliferation of HUVECs through detecting the expression of vascular endothelial growth factor (VEGF) and its receptor (KDR), and the protein expression in the Ras-Raf-MEK-ERK-signalling pathway.Materials and methods HepG2 cell cultural supernatant was used to culture HUVECs to make promote abnormal proliferation, and HSYA was added into the medium. The expression of VEGF, KDR, c-myc, N-ras and NF-κB1 in abnormal HUVEC was detected by RT-qPCR and ELISA at the mRNA and protein levels. Protein expression of ERK signal pathway was measured by Western blot.Results Compared with the abnormal proliferation of HUVECs without any treatment, HSYA inhibited the expression of VEGF and KDR in vitro. Similarly, the protein expression of Ras, p-raf, p-ERK and p-p38MARK in the abnormal HUVECs was reduced when they were treated by HSYA, especially in p-ERK, yet the total raf, ERK, p38MAPK and Akt were not changed whether HSYA existed or not. HSYA could also inhibit the expression of c-myc, N-ras, and NF-κB1.Conclusion When the abnormal HUVECs were treated with HSYA, the low expression of VEGF and KDR reduced the expression of oncogene and transcription factor through the Ras-Raf-MEK-ERK1/2 pathway of the MAPK family. This resulted in inhibiting the abnormal proliferation of HUVECs and angiogenesis.

Experimental acute lung injury induces multi-organ epigenetic modifications in key angiogenic genes implicated in sepsis-associated endothelial dysfunction.

IntroductionThe Tie2/angiopoietin (Tie2/Ang) and vascular endothelial growth factor receptor-ligand systems (VEGFR/VEGF) are recognized to play important roles in the regulation of microvascular endothelial function. Downregulation of these genes during sepsis has been implicated in the pathogenesis of sepsis-related microvascular leak and multiple organ dysfunction syndrome. Mechanisms responsible for dysregulation of angiogenic genes in sepsis are poorly defined.MethodsWestern blot, reverse transcription-polymerase chain reaction, and multiplex chromatin immunoprecipitation platform (Matrix ChIP) were used to investigate serum albumin leak, changes in gene expression, and associated epigenetic alterations in a murine model of acute lung injury-induced sepsis (ALI-sepsis).ResultsExperimental ALI-sepsis induced microvascular leak and downregulation of expression of Angpt1 (Ang1), Tek (Tie2), and Kdr (Vegfr2 or Flk-1) genes in the lung, kidney, and liver. These changes correlate with a decrease in RNA polymerase II density at these genes, and the greatest response was observed in the lung. ALI-sepsis reduced levels of transcription-permissive histone H3 lysine acetylation (H3KAc) at these loci in all examined tissues. Decreases in permissive H3K4m3 and H3Km2 marks were detected only in the lung. In contrast, only minimal alterations in transcription-repressive histone modifications (H3K27m3, H3K9m2, H3K9m3, and H4K20m3) were observed in all tissues.ConclusionsOur results demonstrate that decreases in transcription-permissive, but not increases in transcription-repressive, histone modifications at Angpt1, Tek, and Kdr are a systemic, rather than a lung-restricted, response, involving key end-organs in experimental ALI-sepsis. Given that ventilator-associated pneumonia is a major cause of sepsis in critically ill patients, elucidation of mechanisms mediating epigenetic alterations during sepsis provides fundamental new insights into the pathogenesis of sepsis-induced microvascular leak and subsequent end-organ injury/dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0943-4) contains supplementary material, which is available to authorized users.

VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance

Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo.

Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment

A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.

Construction and measuring combination of KDR-targeted ultrasound contrast agent in vitro for evaluating endometrial receptivity

Objective: To investigate the preparation of a new kind of targeted lipid ultrasound contrast agent with anti-KDR antibody based on biotin- avidin bridge (MB-BAB-KDR) which could combine specifically with KDR that increases during the time of embryo implantation. Then its binding capability in vitro was evaluated. Materials and Methods: The agitation of high-speed method was employed to prepare biotin-microbubbles (MB-B), and biotin–avidin mediated technique was used to produce MB-BAB-KDR. MB-BAB-KDR, MB-B, and biotin- microbubbles-KDR (MB-B-KDR) were incubated with fluorescein-conjugated affiniPure goat anti-rat IgG (H+L) to assess the linked condition. Second, MB-BAB-KDR and control groups (MB-B and MB-B-KDR) were incubated with human umbilical vein endothelial cell (HUVEC). Rosette formation rate was observed and calculated. Then, the parallel plate flow chamber technology was used to access attachment efficiency to KDR Fc. Results: The surface of bubbles could carry KDR antibody through “biotin-avidin” bridge. After incubated with second antibody, bright green fluorescence (II grade) could be observed in MB-BAB-KDR group, as compared with weak fluorescence in control groups of MB-B (0 grade) and MB-B-KDR (I grade). The surrounding rosette formation rate on HUVEC was 89.86% in MB-BAB-KDR group and that of control groups were 7.13% (MB-B-KDR) and 3.02% (MB-B) (p p Conclusion: The successful preparation of MB-BAB-KDR with anti-KDR antibody which shows specially targeting binding capability with HUVEC and stability in shear stress may be served as a noninvasive detection of endometrial vascular KDR expression and provide an experimental foundation for evaluating endometrial receptivity in vivo.

Large platelets but not putative endothelial progenitor cells are associated with low strut coverage after drug-eluting stent implantation

Objectives: This study assessed whether different subsets of circulating endothelial and putative endothelial progenitor cells (CEC and EPC) correlate with stent strut coverage (SSC) using second generation optical coherence tomography (OCT). Background: Due to the lack of imaging modalities with a resolution down to the magnitude of a few cells, the influence of EPC on endothelialisation of drug-eluting stents has not been assessed in patients. Methods: In 37 patients, SSC of everolimus-eluting stents was assessed by OCT 5–7months after stent implantation. Different subsets of EPC (CD34+KDR+, CD34+KDR+CD45dim, CD133+, CD3+CD31+), CEC (CD31+CD45−CD146+), and CD31+CD45−CD146− representing large platelets were analysed by flow cytometry, including viability analyses with 7-AAD. Statistical analysis comprised univariate regression analysis and multivariable models integrating OCT and flow cytometry data as well as clinical variables. Results: SSC and frequency of different cell types were highly comparable with previously published data. EPC defined in part by KDR expression were mostly non-viable. On univariate and in multivariable models, no association between EPC counts and strut coverage was detected. For CD31+CD45−CD146− counts, representing large platelets, an inverse relationship with strut coverage was identified by a multivariable regression model adjusting for age, sex, diabetes mellitus, NYHA and CCS class, CRP, serum triglycerides, glucose and creatinine (beta=−9.42, p=0.006). Conclusions: There was no significant association between EPC or CEC and healing after drug-eluting stent implantation. Yet, CD31+CD45−CD146− cells were associated with low SSC. These data suggest that large platelets may represent a more important mediator of late stent endothelialisation than EPC.

Prostanoids regulate angiogenesis acting primarily on IP and EP4 receptors

Angiogenesis is regulated by numerous activators and inhibitors, including prostanoids. Although many studies have identified their roles in inflammation, regulatory functions of prostanoids in angiogenesis are poorly understood. Here, we compared the activation of angiogenesis in vitro by two prostanoids with important vascular roles: prostaglandin E2 (PGE2) – thought to be the most important prostanoid activator of angiogenesis – and prostaglandin I2 (prostacyclin or PGI2), whose receptors are predominantly expressed in endothelial cells. Both of these prostanoids activate G-protein coupled receptors: EP1, EP2, EP3 and EP4 by PGE2 and IP by prostacyclin. Human umbilical vein endothelial cells (HUVECs) were used to characterize two pivotal pro-angiogenic processes in vitro: cell migration (using the matrigel droplet assay developed in our laboratory) and “tube formation” (a widely accepted method of assessing formation of blood vessel precursors). The suppression of cell migration and tube formation by the IP-specific antagonist CAY10441 was more extensive (~80%) than by the EP4-specific antagonist L-161,982 (~20%). AH6809, an antagonist of EP1, EP2 and EP3 receptors did not significantly suppress angiogenesis. Expression of the pro-angiogenic receptors KDR and Tie-2 in HUVECs was preferentially suppressed by antagonism of IP and EP4 receptors, respectively. EP4 and IP receptor agonists elicited biphasic actions on angiogenic processes in which there was activation at low concentration, and rapid desensitization at high concentrations — a characteristic common to many G-protein coupled receptors. Together these findings suggest that the prostacyclin-IP pathway plays a major role in the regulation of pro-angiogenic processes in HUVECs.

Prognostic significance of combining VEGFA, FLT1 and KDR mRNA expression in lung cancer.

Angiogenesis is important in cancer progression. Promising results in clinical trials have indicated that targeting vascular epidermal growth factor (VEGF) signaling may prolong lung cancer patient survival. In particular, various studies have implicated VEGFA as a potential prognostic marker in lung cancer, although prognostication using the expression of VEGF receptors (VEGFRs), such as fms-related tyrosine kinase 1 (FLT1; also known as VEGFR1) and kinase insert domain receptor (KDR; also known as VEGFR2), has produced varied results in different lung cancer studies. The present study aimed to investigate the prognostic significance of these three factors, alone or in combination. mRNA expression data were extracted from four independent lung cancer cohorts totaling 583 patients, and the association between mRNA expression and survival was investigated by performing statistical analyses. When VEGFA, FLT1 and KDR expression were considered alone, only VEGFA demonstrated a significant association with patient survival consistently across all four datasets (P<0.05). Patients with a high expression of VEGFA and one of the two receptors were associated with significantly worse survival than patients expressing low levels of VEGFA and the particular receptor (P<0.05). Notably, patients with a high level expression of all three genes in their tumor specimens were associated with a significantly shorter survival time compared with patients exhibiting a low level expression of one, two or all three genes (P<0.05). The results indicate that a high level of VEGFA expression and its receptors may be required for cancer progression. Therefore, these three factors should be considered together as a prognostic indicator for lung cancer patients.

Post-infarct treatment with [Pyr1]apelin-13 improves myocardial function by increasing neovascularization and overexpression of angiogenic growth factors in rats

Ischemic heart disease is the leading cause of mortality in the world. Angiogenesis is important for cardiac repair after myocardial infarction (MI) as restores blood supply to the ischemic myocardium and preserves cardiac function. Apelin is a peptide that has been recently shown to potentiate angiogenesis. The aim of this study was to investigate angiogenic effects of [Pyr1]apelin-13 in the rat model of post-MI. Male Wistar rats (n=36) were randomly divided into three groups: (1) sham (2) MI and (3) MI treated with [Pyr1]apelin-13 (MI+Apel). MI animals were subjected to 30min left anterior descending coronary artery (LAD) ligation and 14 days of reperfusion. Twenty-four hours after LAD ligation, [Pyr1]apelin-13 (10nmol/kg/day) was administered i.p. for 5 days. Hemodynamic functions by catheter introduced into the left ventricle (LV), myocardial fibrosis by Masson׳s trichrome staining, gene expression of vascular endothelial growth factor-A (VEGFA), VEGF receptor-2 (Kdr), Ang-1 (angiopoietin-1), Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2) and eNOS by Real-time polymerase chain reaction (Real-Time PCR) and myocardial angiogenesis by CD31 imunostaining were assessed at day 14 post-MI. Post-infarct treatment with [Pyr1]apelin-13 improved LV function and decreased myocardial fibrosis. [Pyr1]apelin-13 treatment led to a significant increase in the expression of VEGFA, Kdr, Ang-1, Tie2 and eNOS. Further, treatment with [Pyr1]apelin-13 promoted capillary density. [Pyr1]apelin-13 has angiogenic and anti-fibrotic activity via formation of new blood vessels and overexpression of VEGFA, Kdr, Ang-1, Tie2 and eNOS in the infarcted myocardium which could in turn repair myocardium and improve LV function.

Cleaved Flt1 ectodomain antagonizes VEGF‐A signaling while uncleaved Flt1 facilitates KDR signaling

Flt1 and KDR are membrane-bound tyrosine kinase receptors that can bind VEGF-A as homo or heterodimers and regulate angiogenesis and vasculogenesis. Flt1 has a 10-fold higher affinity for VEGF-A and brings VEGF-A in proximity to KDR to regulate the effects of VEGF-A on KDR signaling. We have shown that Flt1 is proteolytically cleaved to release an ectodomain into the extracellular milieu which can inhibit VEGF-A induced angiogenesis. We co-expressed Flt1 and KDR in HEK293 cells and studied their interaction, Flt1 cleavage and KDR function. Increasing KDR expression reduces Flt1 ectodomain cleavage and enhances the abundance of uncleaved Flt1 in cell lysates. KDR overexpression stimulates sFlt1 secretion into the extracellular space and progressively reduces free VEGF-A. Co-expression of Flt1 in the presence of KDR increases downstream signaling over that seen with KDR alone. Flt1 ligands, VEGF-A and PLGF reduces ectodomain cleavage of Flt1 where as VEGF-E, a KDR-specific ligand does not. These results suggest that uncleaved Flt1 bound to KDR competes with extracellular sFlt1 and increases receptor activation. Our data also suggests that ligand binding promotes receptor activation by reducing ectodomain receptor cleavage. NIDDK

Determination of Early and Late Endothelial Progenitor Cells in Peripheral Circulation and Their Clinical Association with Coronary Artery Disease.

The clinical implications of early and late endothelial progenitor cells (EPCs) in coronary artery disease (CAD) remain unclear. We investigated endothelial dysfunction in CAD by simultaneously examining early and late EPC colony formation and gene expression of specific surface markers in EPCs. EPCs were extracted from a total of 83 subjects with (n = 47) and without (n = 36) CAD. Early and late EPC colonies were formed from mononuclear cells extracted from peripheral blood. We found that fewer early EPC colonies were produced in the CAD group (7.2 ± 3.l/well) than those in the control group (12.4 ± 1.4/well, p < 0.05), and more late EPC colonies were produced in the CAD group (0.8 ± 0.2/well) than those in the control group (0.25 ± 0.02/well, p < 0.05). In the CAD group, the relative expression of CD31 and KDR of early and late EPCs was lower than in the control group. These results demonstrate that CAD patients could have increased late EPC density and that early and late EPCs in CAD patients exhibited immature endothelial characteristics. We suggest that changes in EPC colony count and gene expression of endothelial markers may have relation with development of CAD.

Left ventricular mass and progenitor cells in chronic heart failure patients.

The aim of the study was to evaluate the association between circulating (CPCs) and endothelial (EPCs) progenitor cells and left ventricular (LV) remodeling in chronic heart failure (HF). 85 HF patients, ranging 29-89 years, 83.5% males, 45.9% ischemic, NYHA functional class II-IV, with a LV ejection fraction ≤40% were studied. LV ejection fraction, LV end-diastolic and end-systolic (LVESV) volumes, LV mass and tricuspid annular plane systolic excursion (TAPSE) were evaluated, and, when indicated, indexed for body surface area (BSA). CPCs and EPCs number was assessed using flow cytometry. CPCs were defined as CD34+, CD133+ and CD34+/CD133+. EPCs, identified through their expression of KDR, were defined as CD34+/KDR+, CD133+/KDR+ and CD34+/CD133+/KDR+. All EPCs were negatively related to LVESV/BSA (r = -0.24, p = 0.02 for all EPC's populations), and to LVmass/BSA (CD34+KDR+; r = -0.30, p = 0.005; CD133+KDR+; r = -0.31, p = 0.004; CD34+CD133+KDR+; r = -0.29, p = 0.007). No differences in EPCs levels in relation to cardiovascular risk factors, medications, etiology, age or gender were observed. CPCs number was higher in women, and lower in ischemic patients. In logistic regression analyses, the low EPCs' number was associated with an increased likelihood of abnormal LVmass/BSA. CPCs proved to be higher and EPCs lower in patients with severely abnormal LVmass/BSA (gr/m(2), ≥122 in women and ≥149 in men). Our results suggest a correlation between LV remodeling and progenitor cells. This is noteworthy considering that it has been suggested that bone marrow-derived EPCs participate in cardiac regeneration and function recovery in the setting of progressive HF.

Induction of Angiogenesis in Zebrafish Embryos and Proliferation of Endothelial Cells by an Active Fraction Isolated from the Root of Astragalus membranaceus using Bioassay-guided Fractionation

The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 3H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 ± 0.27) at 0.125 mg/ml (P < 0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), 3H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells in vitro.

VEGFR tyrosine kinase inhibitor II (VRI) induced vascular insufficiency in zebrafish as a model for studying vascular toxicity and vascular preservation

In ischemic disorders such as chronic wounds and myocardial ischemia, there is inadequate tissue perfusion due to vascular insufficiency. Besides, it has been observed that prolonged use of anti-angiogenic agents in cancer therapy produces cardiovascular toxicity caused by impaired vessel integrity and regeneration. In the present study, we used VEGFR tyrosine kinase inhibitor II (VRI) to chemically induce vascular insufficiency in zebrafish in vivo and human umbilical vein endothelial cells (HUVEC) in vitro to further study the mechanisms of vascular morphogenesis in these pathological conditions. We also explored the possibility of treating vascular insufficiency by enhancing vascular regeneration and repair with pharmacological intervention. We observed that pretreatment of VRI induced blood vessel loss in developing zebrafish by inhibiting angiogenesis and increasing endothelial cell apoptosis, accompanied by down-regulation of kdr, kdrl and flt-1 genes expression. The VRI-induced blood vessel loss in zebrafish could be restored by post-treatment of calycosin, a cardiovascular protective isoflavone. Similarly, VRI induced cytotoxicity and apoptosis in HUVEC which could be rescued by calycosin post-treatment. Further investigation of the underlying mechanisms showed that the PI3K/AKT/Bad cell survival pathway was a main contributor of the vascular regenerative effect of calycosin. These findings indicated that the cardiovascular toxicity in anti-angiogenic therapy was mainly caused by insufficient endothelial cell survival, suggesting its essential role in vascular integrity, repair and regeneration. In addition, we showed that VRI-induced blood vessel loss in zebrafish represented a simple and effective in vivo model for studying vascular insufficiency and evaluating cancer drug vascular toxicities.

Local VEGF inhibition prevents ovarian alterations associated with ovarian hyperstimulation syndrome

The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS) is partially mediated by vascular endothelial growth factor A (VEGF). The aim of this study was to investigate the effects of VEGF inhibition on the development of corpora lutea (CL) and cystic structures, steroidogenesis, apoptosis, cell proliferation, endothelial cell area, VEGF receptors (KDR and Flt-1), claudin-5 and occludin levels in ovaries from an OHSS rat model. The VEGF inhibitor used (VEGF receptor-1 (FLT-1)/Fc chimera, TRAP) decreased the concentrations of progesterone and estradiol as well as the percentage of CL and cystic structures in OHSS rats, and increased apoptosis in CL. Endothelial cell area in CL and KDR expression and its phosphorylation were increased, whereas claudin-5 and occludin levels were decreased in the OHSS compared to the control TRAP reversed these parameters. Our findings indicate that VEGF inhibition prevents the early onset of OHSS and decreases its severity in rats.

Unique origin of eye canal combines elements of blood, lymph tube development.

Sometimes things aren't either-or, but a little of both. That turns out to be the case for Schlemm's canal (Figure 1), a tiny but vital structure that drains fluid from the eye, keeping it healthy and preventing the build-up of pressure that can lead to glaucoma. Using an innovative combination of approaches, Krishnakumar Kizhatil, Simon John, and colleagues show that the development of Schlemm's canal is a unique process that shares features with the formation of both blood vessels and lymph vessels. Figure 1 Schlemm's canal (cyan) is a key vessel that drains a clear fluid (aqueous humor) from the eye. The fluid drains into blood vessels (magenta) that are connected to the canal. First, a bit of ocular anatomy. The eye's outer layer (the “ocular wall”) includes the sclera and, at the frontmost portion, the cornea. Behind the cornea is the fluid-filled anterior chamber. At the rear of the chamber sits the iris (with the lens just behind that). At its circumference, the iris meets the ocular wall. Just at this juncture, embedded within the ocular wall and encircling the edge of the disc-shaped anterior chamber, sits Schlemm's canal. Schlemm's canal drains the fluid within the chamber, called aqueous humor, and regulates the passage of immune cells exiting the eye into the bloodstream. The details of the development of the canal have been controversial, in large part because it has been difficult to image the developing canal with sufficient clarity to follow the cellular fates of structures potentially involved. The authors overcame this problem by developing a method for observing under the microscope the entire front portion of the eye, out to the border between the cornea and sclera (the limbus), including Schlemm's canal. Working in mice, this technique allowed them to study its development in unprecedented three-dimensional detail and to apply lineage-specific markers to determine the origin of cells making up the canal. Prominent among their findings was that cells of the canal express not only characteristic blood vessel markers but also Prox1, a master regulator of lymphatic phenotypes. This suggested that canal cells might derive from lymphatic vessels, but by labeling and following cells expressing a second lymphatic marker, they ruled out a lymphatic origin for canal cells. Similarly, they were not derived from neural crest cells. Instead, the developmental program of canal cells appears to combine elements of angiogenesis, lymphangiogenesis, and vasculogenesis. Detailed microscopic analysis indicated that canal cells derived from blood vessels of the limbus. Here, the authors found two vascular beds, oriented at right angles to each other, sandwiching the mature canal. During development, they found, the endothelial cells in each sprouted so-called tip cells, previously seen in development of new blood vessels from existing ones. In canal development, the authors observed tip cells growing out from their parent vessels, eventually clustering to form a continuous chain of cells around the limbus—the beginning of the canal. Over the ensuing days, Prox-1 expression increased, inner and outer walls of the canal began to develop, and finally, the lumen began to form. The authors show that expression of KDR, a receptor for vascular endothelial growth factor, was essential to the process; however, most of the genetic and cell-signaling details governing this fascinating process remain to be worked out. Further work to understand canal formation will be greatly aided by the methods developed in this study. Understanding the details of canal development may lead to a better understanding of the working of the canal and how it can be manipulated to control glaucoma. Kizhatil K, Ryan M, Marchant JK, Henrich S, John SWM (2014) Schlemm's Canal Is a Unique Vessel with a Combination of Blood Vascular and Lymphatic Phenotypes that Forms by a Novel Developmental Process. doi:10.1371/journal.pbio.1001912

Mammalian lysine histone demethylase KDM2A regulates E2F1-mediated gene transcription in breast cancer cells.

It is established that histone modifications like acetylation, methylation, phosphorylation and ubiquitination affect chromatin structure and modulate gene expression. Lysine methylation/demethylation on Histone H3 and H4 is known to affect transcription and is mediated by histone methyl transferases and histone demethylases. KDM2A/JHDM1A/FBXL11 is a JmjC-containing histone demethylase that targets mono- and dimethylated Lys36 residues of Histone H3; its function in breast cancer is not fully understood. Here we show that KDM2A is strongly expressed in myoepithelial cells (MEPC) in breast cancer tissues by immunohistochemistry. Ductal cells from ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) show positive staining for KDM2A, the expression decreases with disease progression to metastasis. Since breast MEPCs have tumor-suppressive and anti-angiogenic properties, we hypothesized that KDM2A could be contributing to some of these functions. Silencing KDM2A with small interfering RNAs demonstrated increased invasion and migration of breast cancer cells by suppressing a subset of matrix metalloproteinases (MMP-2, -9, -14 and -15), as seen by real-time PCR. HUVEC cells showed increased angiogenic tubule formation ability in the absence of KDM2A, with a concomitant increase in the expression of VEGF receptors, FLT-1 and KDR. KDM2A physically bound to both Rb and E2F1 in a cell cycle dependent manner and repressed E2F1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assays revealed that KDM2A associates with E2F1-regulated proliferative promoters CDC25A and TS in early G-phase and dissociates in S-phase. Further, KDM2A could also be detected on MMP9, 14 and 15 promoters, as well as promoters of FLT1 and KDR. KDM2A could suppress E2F1-mediated induction of these promoters in transient transfection experiments. These results suggest a regulatory role for KDM2A in breast cancer cell invasion and migration, through the regulation of E2F1 function.

The angiogenic behaviors of human umbilical vein endothelial cells (HUVEC) in co-culture with osteoblast-like cells (MG-63) on different titanium surfaces

ObjectivesInteraction between osteogenesis and angiogenesis plays an important role in implant osseointegration. In the present study we investigated the influence of titanium surface properties on the angiogenic behaviors of endothelial cells grown in direct contact co-culture with osteoblasts. MethodsHuman umbilical vein endothelial cells (HUVECs) and osteoblast-like cells (MG-63 cells) were grown in direct co-culture on the following titanium surfaces: acid-etched (A), hydrophilic A (modA), coarse-gritblasted and acid-etched (SLA) and hydrophilic SLA (SLActive). Cell proliferation was evaluated by cell counting combined with flow cytometry. The expression of von Willebrand Factor (vWF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), E-Selectin, as well as vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR in HUVECs and VEGF in MG-63 were measured by qPCR. The dynamic behavior of endothelial cells was recorded by time-lapse microscopy. ResultsProliferation of HUVECs was highest on A, followed by SLA, modA and SLActive surfaces. The expression of vWF, TM, EPCR, E-Selectin and Flt-1 in HUVECs was significantly higher on A than on all other surfaces. The expression of KDR in HUVECs grown on A surface was below detection limit. VEGF expression in MG-63 cells was significantly higher on SLActive vs SLA and modA vs A surfaces. Time-lapse microscopy revealed that HUVECs moved quickest and formed cell clusters earlier on A surface, followed by SLA, modA and SLActive surface. ConclusionsIn co-culture conditions, proliferation and expression of angiogenesis associated genes in HUVECs are promoted by smooth hydrophobic Ti surface, which is in contrast to previous mono-culture studies.

Active angiogenesis in metastatic renal cell carcinoma predicts clinical benefit to sunitinib-based therapy.

Background:Sunitinib represents a widely used therapy for metastatic renal cell carcinoma patients. Even so, there is a group of patients who show toxicity without clinical benefit. In this work, we have analysed pivotal molecular targets involved in angiogenesis (vascular endothelial growth factor (VEGF)-A, VEGF receptor 2 (KDR), phosphorylated (p)KDR and microvascular density (MVD)) to test their potential value as predictive biomarkers of clinical benefit in sunitinib-treated renal cell carcinoma patients.Methods:Vascular endothelial growth factor-A, KDR and pKDR-Y1775 expression as well as CD31, for MVD visualisation, were determined by immunohistochemistry in 48 renal cell carcinoma patients, including 23 metastatic cases treated with sunitinib. Threshold was defined for each biomarker, and univariate and multivariate analyses for progression-free survival (PFS) and overall survival (OS) were carried out.Results:The HistoScore mean value obtained for VEGF-A was 121.6 (range, 10–300); for KDR 258.5 (range, 150–300); for pKDR-Y1775 10.8 (range, 0–65) and the mean value of CD31-positive structures for MVD visualisation was 49 (range, 10–126). Statistical differences for PFS (P=0.01) and OS (P=0.007) were observed for pKDR-Y1775 in sunitinib-treated patients. Importantly, pKDR-Y1775 expression remained significant after multivariate Cox analysis for PFS (P=0.01; HR: 5.35, 95% CI, 1.49–19.13) and for OS (P=0.02; HR: 5.13, 95% CI, 1.25–21.05).Conclusions:Our results suggest that the expression of phosphorylated (i.e., activated) KDR in tumour stroma might be used as predictive biomarker for the clinical outcome in renal cell carcinoma first-line sunitinib-treated patients.

Endoglin, VEGF, and its receptors in predicting metastases in endometrial carcinoma

Preoperative evaluation of the risk for metastases in endometrial carcinoma is challenging. The growth of new vessels, angiogenesis, is important for tumor growth and purported to be involved in the metastatic process. The aim of this study was to evaluate the significance of preoperative serum levels and immunohistochemical expression of angiogenic markers in predicting a metastasized disease. Preoperative sera from 98 consecutive women presenting with endometrial carcinoma were collected. Serum concentrations of VEGF, sFLT1, and CD105 were assessed by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to assess the expression of CD105, VEGF, FLT1, and KDR. The results were correlated to the presence of metastases, presence of deep (≥50%) myometrial invasion, and the histological grade of the tumor. Tumors with other than endometrioid histology were excluded. Of the 80 evaluable patients, 11 had a metastasized disease. The serum concentration of VEGF was higher in the group with metastases than in the group without metastases (median [range], 743 pg/mL [546-1,183 pg/mL] vs. 383 pg/mL [31-1,524 pg/mL], p < 0.001, respectively). In the multivariable analysis, the concentration of VEGF was the sole independent, albeit weak predictive factor for the presence of metastases (odds ratio, 1.004, 95% confidence interval, 1.002-1.007; p = 0.001). The immunohistochemical expression of the markers was not associated with any of the clinicopathological features of the tumors. The results of the present study suggest that preoperative serum VEGF concentration correlates with the presence of metastases in endometrioid endometrial carcinoma.