Identification of New Markers of Angiogenic Sprouting Using Transcriptomics: New Role for RND3.

BACKGROUND: New blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. METHODS: Using a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from noninvading cells. We used this model to perform multiple transcriptomics analyses from independent donors to monitor endothelial gene expression changes. RESULTS: Single-cell population analyses, single-cell cluster analyses, and bulk RNA sequencing revealed common transcriptomic changes associated with invading cells. We also found that collagenase digestion used to isolate single cells upregulated the Fos proto-oncogene transcription factor. Exclusion of Fos proto-oncogene expressing cells revealed a gene signature consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis and included multiple tip cell markers. Upregulation of SNAI1 (snail family transcriptional repressor 1), PTGS2 (prostaglandin synthase 2), and JUNB (JunB proto-oncogene) protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated rounding 3, also known as RhoE, which has not yet been implicated in angiogenesis. Silencing rounding 3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for rounding 3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared with wild-type littermates. CONCLUSIONS: Validation of multiple genes, including rounding 3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

TWIST1 and TSG6 are coordinately regulated and function as potency biomarkers in human MSCs.

Mesenchymal stem/stromal cells (MSCs) have been evaluated in >1500 clinical trials, but outcomes remain suboptimal because of knowledge gaps in quality attributes that confer potency. We show that TWIST1 directly represses TSG6 expression that TWIST1 and TSG6 are inversely correlated across bone marrow-derived MSC (BM-MSC) donor cohorts and predict interdonor differences in their proangiogenic, anti-inflammatory, and immune suppressive activity in vitro and in sterile inflammation and autoimmune type 1 diabetes preclinical models. Transcript profiling of TWIST1HiTSG6Low versus TWISTLowTSG6Hi BM-MSCs revealed previously unidentified roles for TWIST1/TSG6 in regulating cellular oxidative stress and TGF-ß2 in modulating TSG6 expression and anti-inflammatory activity. TWIST1 and TSG6 levels also correlate to donor stature and predict differences in iPSC-derived MSC quality attributes. These results validate TWIST1 and TSG6 as biomarkers that predict interdonor differences in potency across laboratories and assay platforms, thereby providing a means to manufacture MSC products tailored to specific diseases.

Perfusability and immunogenicity of implantable pre-vascularized tissues recapitulating features of native capillary network.

Vascularization is a key pre-requisite to engineered anatomical scale three dimensional (3-D) constructs to ensure their nutrient and oxygen supply upon implantation. Presently, engineered pre-vascularized 3-D tissues are limited to only micro-scale hydrogels, which meet neither the anatomical scale needs nor the complexity of natural extracellular matrix (ECM) environments. Anatomical scale perfusable constructs are critically needed for translational applications. To overcome this challenge, we previously developed pre-vascularized ECM sheets with long and oriented dense microvascular networks. The present study further evaluated the patency, perfusability and innate immune response toward these pre-vascularized constructs. Macrophage-co-cultured pre-vascularized constructs were evaluated in vitro to confirm micro-vessel patency and perturbations in macrophage metabolism. Subcutaneously implanted pre-vascularized constructs remained viable and formed a functional anastomosis with host vasculature within 3 days of implantation. This completely biological pre-vascularized construct holds great potential as a building block to engineer perfusable anatomical scale tissues.

Conjugated Bile Acids Promote Lymphangiogenesis by Modulation of the Reactive Oxygen Species-p90RSK-Vascular Endothelial Growth Factor Receptor 3 Pathway.

Conjugated bile acids (BA) are significantly elevated in several liver pathologies and in the metastatic lymph node (LN). However, the effects of BAs on pathological lymphangiogenesis remains unknown. The current study explores the effects of BAs on lymphangiogenesis. BA levels were elevated in the LN and serum of Mdr2-/- mice (model of sclerosing cholangitis) compared to control mice. Liver and LN tissue sections showed a clear expansion of the lymphatic network in Mdr2-/- mice, indicating activated lymphangiogenic pathways. Human lymphatic endothelial cells (LECs) expressed BA receptors and a direct treatment with conjugated BAs enhanced invasion, migration, and tube formation. BAs also altered the LEC metabolism and upregulated key metabolic genes. Further, BAs induced the production of reactive oxygen species (ROS), that in turn phosphorylated the redox-sensitive kinase p90RSK, an essential regulator of endothelial cell dysfunction and oxidative stress. Activated p90RSK increased the SUMOylation of the Prox1 transcription factor and enhanced VEGFR3 expression and 3-D LEC invasion. BA-induced ROS in the LECs, which led to increased levels of Yes-associated protein (YAP), a lymphangiogenesis regulator. The suppression of cellular YAP inhibited BA-induced VEGFR3 upregulation and lymphangiogenic mechanism. Overall, our data shows the expansion of the lymphatic network in presclerotic liver disease and establishes a novel mechanism whereby BAs promote lymphangiogenesis.

Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation.

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild-type endothelial cells mimicked the ANXA2 knock-down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events.

MicroRNA-150 (miR-150) and Diabetic Retinopathy: Is miR-150 Only a Biomarker or Does It Contribute to Disease Progression?

Diabetic retinopathy (DR) is a chronic disease associated with diabetes mellitus and is a leading cause of visual impairment among the working population in the US. Clinically, DR has been diagnosed and treated as a vascular complication, but it adversely impacts both neural retina and retinal vasculature. Degeneration of retinal neurons and microvasculature manifests in the diabetic retina and early stages of DR. Retinal photoreceptors undergo apoptosis shortly after the onset of diabetes, which contributes to the retinal dysfunction and microvascular complications leading to vision impairment. Chronic inflammation is a hallmark of diabetes and a contributor to cell apoptosis, and retinal photoreceptors are a major source of intraocular inflammation that contributes to vascular abnormalities in diabetes. As the levels of microRNAs (miRs) are changed in the plasma and vitreous of diabetic patients, miRs have been suggested as biomarkers to determine the progression of diabetic ocular diseases, including DR. However, few miRs have been thoroughly investigated as contributors to the pathogenesis of DR. Among these miRs, miR-150 is downregulated in diabetic patients and is an endogenous suppressor of inflammation, apoptosis, and pathological angiogenesis. In this review, how miR-150 and its downstream targets contribute to diabetes-associated retinal degeneration and pathological angiogenesis in DR are discussed. Currently, there is no effective treatment to stop or reverse diabetes-caused neural and vascular degeneration in the retina. Understanding the molecular mechanism of the pathogenesis of DR may shed light for the future development of more effective treatments for DR and other diabetes-associated ocular diseases.

Hypertensive Stimuli Indirectly Stimulate Lymphangiogenesis through Immune Cell Secreted Factors.

(1) Background: Renal immune cells and lymphatic vessel (LV) density have been reported previously to be increased in multiple mouse models of hypertension (HTN). However, whether interstitial levels of HTN stimuli such as angiotensin II, salt, or asymmetric dimethylarginine have a direct or indirect effect on lymphangiogenesis is unknown. We hypothesized that these 3 HTN stimuli directly increase lymphatic endothelial cell (LEC) proliferation, LEC 3-D matrix invasion and vessel formation, and sprouting of mouse mesometrial LVs. (2) Methods: Human LECs (hLECs) and mouse LECs (mLECs) were treated with HTN stimuli while explanted mouse mesometrial LVs were treated with either the same HTN stimuli or with HTN stimuli-conditioned media. Conditioned media was prepared by treating murine splenocytes with HTN stimuli. (3) Results: HTN stimuli had no direct effect on hLEC or mLEC proliferation. Treatment of hLECs with HTN stimuli increased the number of lumen-forming structures and invasion distance (both p < 0.05) in the 3-D matrix but decreased the average lumen diameter and the number of cells per invading structure (both p < 0.05). Conditioned media from HTN-stimuli-treated splenocytes significantly attenuated the decrease in sprout number (aside from salt) and sprout length of mouse mesometrial LVs that is found in the HTN stimuli alone. (4) Conclusions: These data indicate that HTN stimuli indirectly prevent a decrease in lymphangiogenesis through secreted factors from HTN-stimuli-treated immune cells.

EZH2 and Endometrial Cancer Development: Insights from a Mouse Model.

Enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive complex 2, plays an important role in cancer development. As both oncogenic and tumor suppressive functions of EZH2 have been documented in the literature, the objective of this study is to determine the impact of Ezh2 deletion on the development and progression of endometrial cancer induced by inactivation of phosphatase and tensin homolog (PTEN), a tumor suppressor gene frequently dysregulated in endometrial cancer patients. To this end, we created mice harboring uterine deletion of both Ezh2 and Pten using Cre recombinase driven by the progesterone receptor (Pgr) promoter. Our results showed reduced tumor burden in Ptend/d; Ezh2d/d mice compared with that of Ptend/d mice during early carcinogenesis. The decreased Ki67 index in EZH2 and PTEN-depleted uteri versus that in PTEN-depleted uteri indicated an oncogenic role of EZH2 during early tumor development. However, mice harboring uterine deletion of both Ezh2 and Pten developed unfavorable disease outcome, accompanied by exacerbated epithelial stratification and heightened inflammatory response. The observed effect was non-cell autonomous and mediated by altered immune response evidenced by massive accumulation of intraluminal neutrophils, a hallmark of endometrial carcinoma in Ptend/d; Ezh2d/d mice during disease progression. Hence, these results reveal dual roles of EZH2 in endometrial cancer development.

Tumor Lymphatic Interactions Induce CXCR2-CXCL5 Axis and Alter Cellular Metabolism and Lymphangiogenic Pathways to Promote Cholangiocarcinoma.

Cholangiocarcinoma (CCA), or cancer of bile duct epithelial cells, is a very aggressive malignancy characterized by early lymphangiogenesis in the tumor microenvironment (TME) and lymph node (LN) metastasis which correlate with adverse patient outcome. However, the specific roles of lymphatic endothelial cells (LECs) that promote LN metastasis remains unexplored. Here we aimed to identify the dynamic molecular crosstalk between LECs and CCA cells that activate tumor-promoting pathways and enhances lymphangiogenic mechanisms. Our studies show that inflamed LECs produced high levels of chemokine CXCL5 that signals through its receptor CXCR2 on CCA cells. The CXCR2-CXCL5 signaling axis in turn activates EMT (epithelial-mesenchymal transition) inducing MMP (matrix metalloproteinase) genes such as GLI, PTCHD, and MMP2 in CCA cells that promote CCA migration and invasion. Further, rate of mitochondrial respiration and glycolysis of CCA cells was significantly upregulated by inflamed LECs and CXCL5 activation, indicating metabolic reprogramming. CXCL5 also induced lactate production, glucose uptake, and mitoROS. CXCL5 also induced LEC tube formation and increased metabolic gene expression in LECs. In vivo studies using CCA orthotopic models confirmed several of these mechanisms. Our data points to a key finding that LECs upregulate critical tumor-promoting pathways in CCA via CXCR2-CXCL5 axis, which further augments CCA metastasis.

CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function.

Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G protein-coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine 1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function.

Interleukin 35 Delays Hindlimb Ischemia-Induced Angiogenesis Through Regulating ROS-Extracellular Matrix but Spares Later Regenerative Angiogenesis.

Interleukin (IL) 35 is a novel immunosuppressive heterodimeric cytokine in IL-12 family. Whether and how IL-35 regulates ischemia-induced angiogenesis in peripheral artery diseases are unrevealed. To fill this important knowledge gap, we used loss-of-function, gain-of-function, omics data analysis, RNA-Seq, in vivo and in vitro experiments, and we have made the following significant findings: i) IL-35 and its receptor subunit IL-12RB2, but not IL-6ST, are induced in the muscle after hindlimb ischemia (HLI); ii) HLI-induced angiogenesis is improved in Il12rb2-/- mice, in ApoE-/-/Il12rb2-/- mice compared to WT and ApoE-/- controls, respectively, where hyperlipidemia inhibits angiogenesis in vivo and in vitro; iii) IL-35 cytokine injection as a gain-of-function approach delays blood perfusion recovery at day 14 after HLI; iv) IL-35 spares regenerative angiogenesis at the late phase of HLI recovery after day 14 of HLI; v) Transcriptome analysis of endothelial cells (ECs) at 14 days post-HLI reveals a disturbed extracellular matrix re-organization in IL-35-injected mice; vi) IL-35 downregulates three reactive oxygen species (ROS) promoters and upregulates one ROS attenuator, which may functionally mediate IL-35 upregulation of anti-angiogenic extracellular matrix proteins in ECs; and vii) IL-35 inhibits human microvascular EC migration and tube formation in vitro mainly through upregulating anti-angiogenic extracellular matrix-remodeling proteins. These findings provide a novel insight on the future therapeutic potential of IL-35 in suppressing ischemia/inflammation-triggered inflammatory angiogenesis at early phase but sparing regenerative angiogenesis at late phase.

The ß1-integrin plays a key role in LEC invasion in an optimized 3-D collagen matrix model.

Lymphangiogenesis, or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration, and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro three-dimensional (3-D) collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), interleukin (IL)-8, and hepatocyte growth factor (HGF), were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion, and synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expressions of the integrin α5- and integrin ß1-subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA-mediated knockdown of the integrin ß1-subunit suppressed LEC invasion and also inhibited VEGF receptor (VEGFR)3 and ERK activation. Furthermore, exposing LECs to the inflammatory mediator lipopolysaccharide (LPS) inhibited sprouting. This optimized model for LEC invasion includes S1P, VEGF-C, and fibronectin within a 3-D collagen matrix, along with VEGF-C, VEGF-A, bFGF, and HGF in the culture medium, and provides a useful tool to investigate the functional effect of various lymphangiogenic factors and inhibitors.

Transforming growth factor beta signaling and decidual integrity in mice†.

Transforming growth factor beta (TGFß) signaling regulates multifaceted reproductive processes. It has been shown that the type 1 receptor of TGFß (TGFBR1) is indispensable for female reproductive tract development, implantation, placental development, and fertility. However, the role of TGFß signaling in decidual development and function remains poorly defined. Our objective is to determine the impact of uterine-specific deletion of Tgfbr1 on decidual integrity, with a focus on the cellular and molecular properties of the decidua during development. Our results show that the developmental dynamics of the decidua is altered in TGFBR1 conditionally depleted uteri from embryonic day (E) 5.5 to E8.5, substantiated by downregulation of genes associated with inflammatory responses and uterine natural killer cell abundance, reduced presence of nondecidualized fibroblasts in the antimesometrial region, and altered decidual cell development. Notably, conditional ablation of TGFBR1 results in the formation of decidua containing more abundant alpha smooth muscle actin (ACTA2)-positive cells at the peripheral region of the antimesometrial side versus controls at E6.5-E8.5. This finding is corroborated by upregulation of a subset of smooth muscle marker genes in Tgfbr1 conditionally deleted decidua at E6.5 and E8.5. Moreover, increased cell proliferation and enhanced decidual ERK1/2 signaling were found in Tgfbr1 conditional knockout mice upon decidual regression. In summary, conditional ablation of TGFBR1 in the uterus profoundly impacts the cellular and molecular properties of the decidua. Our results suggest that TGFBR1 in uterine epithelial and stromal compartments is important for the integrity of the decidua, a transient but crucial structure that supports embryo development.

Hypoxic tumor microenvironment: Implications for cancer therapy.

IMPACT STATEMENT: Hypoxia contributes to tumor aggressiveness and promotes growth of many solid tumors that are often resistant to conventional therapies. In order to achieve successful therapeutic strategies targeting different cancer types, it is necessary to understand the molecular mechanisms and signaling pathways that are induced by hypoxia. Aberrant tumor vasculature and alterations in cellular metabolism and drug resistance due to hypoxia further confound this problem. This review focuses on the implications of hypoxia in an inflammatory TME and its impact on the signaling and metabolic pathways regulating growth and progression of cancer, along with changes in lymphangiogenic and angiogenic mechanisms. Finally, the overarching role of hypoxia in mediating therapeutic resistance in cancers is discussed.

Mechanotransduction drives morphogenesis to develop folding during placental development in pigs.

INTRODUCTION: Our aim was to evaluate whether mechanical forces applied to the placenta of pigs correlate with morphological changes that coordinate the development of placental folds. METHODS: We examined changes in the length of placental folds, expression of mechanotransduction-implicated molecules in placental tissues, changes in the size of subepithelial blood vessels within the endometrium, and effects of in vivo supplementation with arginine on fold development. RESULTS: We observed that: 1) the length of folds increased 2) osteopontin, talin and focal adhesion kinase co-localized into aggregates at the maternal placental (uterine)-fetal placental interface; 3) filamin, actin related protein 2, and F-actin were enriched in the tops of maternal placental folds extending into fetal placental tissue; 4) maternal stromal fibroblasts acquired alpha smooth muscle actin; 5) endometrial blood vessels increased in size; and 6) supplementation with arginine increased fold length. CONCLUSION: Results indicate that lengthening of folds associates with polymerization of actin that coincides with FA assembly, endometrial fibroblasts differentiate into myofibroblasts, and dilation of subepithelial blood vessels correlates with development of folds that is enhanced by arginine. We propose that dilation of subepithelial endometrial blood vessels delivers increased blood flow that pushes upward on the interface between the uterine luminal epithelium (LE) and the placental chorionic epithelium (CE), protrusive forces from growing uterine blood vessels trigger focal adhesion assembly and actin polymerization between the LE and CE, and endometrial fibroblasts differentiate into contractile myofibroblasts that pull connective tissue downward and inward to sculpt folds at the maternal placental-fetal placental interface.

OPN binds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature.

Angiogenesis is fundamental to the expansion of the placental vasculature during pregnancy. Integrins are associated with vascular formation; and osteopontin is a candidate ligand for integrins to promote angiogenesis. Endothelial progenitor cells (EPCs) are released from bone marrow into the blood and incorporate into newly vascularized tissue where they differentiate into mature endothelium. Results of studies in women suggest that EPCs may play an important role in maintaining placental vascular integrity during pregnancy, although little is known about how EPCs are recruited to these tissues. Our goal was to determine the αv integrin mediated effects of osteopontin on EPC adhesion and incorporation into angiogenic vascular networks. EPCs were isolated from 6 h old piglets. RT-PCR revealed that EPCs initially had a monocyte-like phenotype in culture that became more endothelial-like with cell passage. Immunofluorescence microscopy confirmed that the EPCs express platelet endothelial cell adhesion molecule, vascular endothelial cadherin, and von Willebrand factor. When EPCs were cultured on OPN-coated slides, the αv integrin subunit was observed in focal adhesions at the basal surface of EPCs. Silencing of αv integrin reduced EPC binding to OPN and focal adhesion assembly. In vitro siRNA knockdown in EPCs,demonstrated that OPN stimulates EPC incorporation into human umbilical vein endothelial cell (HUVEC) networks via αv-containing integrins. Finally, in situ hybridization and immunohistochemistry localized osteopontin near placental blood vessels. In summary, OPN binds the αv integrin subunit on EPCs to support EPC adhesion and increase EPC incorporation into angiogenic vascular networks.

FTY720, a sphingosine analog, altered placentome histoarchitecture in ewes.

BACKGROUND: The lysosphingolipid, sphingosine-1-phosphate, is a well-described and potent pro-angiogenic factor. Receptors, as well as the sphingosine phosphorylating enzyme sphingosine kinase 1, are expressed in the placentomes of sheep and the decidua of rodents; however, a function for this signaling pathway during pregnancy has not been established. The objective of this study was to investigate whether sphingosine-1-phosphate promoted angiogenesis within the placentomes of pregnant ewes. Ewes were given daily jugular injections of FTY720 (2-amino-2[2-(- 4-octylphenyl)ethyl]propate-1,3-diol hydrochloride), an S1P analog. RESULTS: FTY720 infusion from days 30 to 60 of pregnancy did not alter maternal organ weights nor total number or mass of placentomes, but did alter placentome histoarchitecture. Interdigitation of caruncular crypts and cotyledonary villi was decreased, as was the relative area of cotyledonary tissue within placentomes. Also, the percentage of area occupied by cotyledonary villi per unit of placentome was increased, while the thickness of the caruncular capsule was decreased in ewes treated with FTY720. Further, FTY720 infusion decreased the number and density of blood vessels within caruncular tissue near the placentome capsule where the crypts emerge from the capsule. Finally, FTY720 infusion decreased asparagine and glutamine in amniotic fluid and methionine in allantoic fluid, and decreased the crown rump length of day 60 fetuses. CONCLUSIONS: While members of the sphingosine-1-phosphate signaling pathway have been characterized within the uteri and placentae of sheep and mice, the present study uses FTY720 to address the influence of S1P signaling on placental development. We present evidence that modulation of the S1P signaling pathway results in the alteration of caruncular vasculature, placentome architecture, abundance of amino acids in allantoic and amniotic fluids, and fetal growth during pregnancy in sheep. The marked morphological changes in placentome histoarchitecture, including alteration in the vasculature, may be relevant to fetal growth and survival. It is somewhat surprising that fetal length was reduced as early as day 60, because fetal growth in sheep is greatest after day 60. The subtle changes observed in the fetuses of ewes exposed to FTY720 may indicate an adaptive response of the fetuses to cope with altered placental morphology.

Newly Identified Peptide, Peptide Lv, Promotes Pathological Angiogenesis.

Background We recently discovered a small endogenous peptide, peptide Lv, with the ability to activate vascular endothelial growth factor receptor 2 and its downstream signaling. As vascular endothelial growth factor through vascular endothelial growth factor receptor 2 contributes to normal development, vasodilation, angiogenesis, and pathogenesis of various diseases, we investigated the role of peptide Lv in vasodilation and developmental and pathological angiogenesis in this study. Methods and Results The endothelial cell proliferation, migration, and 3-dimensional sprouting assays were used to test the abilities of peptide Lv in angiogenesis in vitro. The chick chorioallantoic membranes and early postnatal mice were used to examine its impact on developmental angiogenesis. The oxygen-induced retinopathy and laser-induced choroidal neovascularization mouse models were used for in vivo pathological angiogenesis. The isolated porcine retinal and coronary arterioles were used for vasodilation assays. Peptide Lv elicited angiogenesis in vitro and in vivo. Peptide Lv and vascular endothelial growth factor acted synergistically in promoting endothelial cell proliferation. Peptide Lv-elicited vasodilation was not completely dependent on nitric oxide, indicating that peptide Lv had vascular endothelial growth factor receptor 2/nitric oxide-independent targets. An antibody against peptide Lv, anti-Lv, dampened vascular endothelial growth factor-elicited endothelial proliferation and laser-induced vascular leakage and choroidal neovascularization. While the pathological angiogenesis in mouse eyes with oxygen-induced retinopathy was enhanced by exogenous peptide Lv, anti-Lv dampened this process. Furthermore, deletion of peptide Lv in mice significantly decreased pathological neovascularization compared with their wild-type littermates. Conclusions These results demonstrate that peptide Lv plays a significant role in pathological angiogenesis but may be less critical during development. Peptide Lv is involved in pathological angiogenesis through vascular endothelial growth factor receptor 2-dependent and -independent pathways. As anti-Lv dampened the pathological angiogenesis in the eye, anti-Lv may have a therapeutic potential to treat pathological angiogenesis.