Normal Angiogenesis Research Articles

Substantial Toxic Effect of Water-Pipe Smoking on the Early Stage of Embryonic Development.

Water-pipe smoking (WPS) is the most widespread tobacco use in the Middle-East, and is rapidly spreading globally. Smoke from WP contains most of the compounds present in cigarette smoke, although in different proportions. WPS is associated with the risk of several human diseases; however, its impact on the early stage of normal development has not been investigated yet. Thus, in this investigation, we assess the effect of WPS on the embryo at the early stage of development. Chicken embryos at 3 days of incubations were used in this study. Meanwhile, we explored the outcome of WPS on angiogenesis using the chorioallantoic membrane (CAM) of the chicken embryos. Finally, quantitative real-time polymerase chain reaction was used to study the regulation of some key control genes of cell proliferation, apoptosis, and migration. Our data reveal that WPS inhibits angiogenesis of the CAM and in embryos in comparison with their matched controls; in addition, WPS-exposed embryos show slight reduction in their sizes. We also noted that around 80% of WPS-exposed embryos die before 10 days of incubation. More significantly, WPS induces upregulations of BCL-2, Caspase-8, ATF-3, INHIB-A, and Cadherin 6 genes, which are important key regulators of cell apoptosis, proliferation, and migration. Our data reveal, for the first time, that WPS has very toxic effects during the early stage of embryogenesis. Thus, we believe that further studies are required to elucidate the pathogenic effect of WPS on human health especially on the embryo at the early stage of its development. This investigation addresses an important gap on the outcome of WPS during the early stage of embryogenesis. Data of this study point out that WPS can have a very toxic effect on the embryo at this stage. Additionally, results from this report display for the first time that WPS can damage normal angiogenesis of the embryo thus provoking a significant number of embryonic death. Moreover, this study reveals that this effect can occur via the deregulation of several genes related to cell apoptosis, proliferation, and migration.

Angiogenesis Research: Extramural Portfolio Supported by the National Heart, Lung, and Blood Institute, 2008-2015.

The National Heart, Lung, and Blood Institute funds angiogenesis-related extramural research aimed at improving the understanding of normal and abnormal angiogenesis and translating mechanistic findings into therapeutic interventions. This portfolio analysis of fiscal years 2008 through 2015 shows that the National Heart, Lung, and Blood Institute awarded an average of 229±26 angiogenesis-related grants with an average cost of $116±20 million per fiscal year. Angiogenesis research was funded through 4 major funding mechanisms, mainly through Research Project Grants (R01s, 64% of the total awards). New awards (type 1) that may represent pursuit of new directions in angiogenesis research accounted for an average of 19±3% of total awards per fiscal year. In the portfolio, 74.1% of awards (65% of total dollars) used in vivo animal models; 9.7% of awards (5% of total dollars) used in vitro methods; 13.7% of awards (27% of total dollars) involved human subject studies (not including clinical trials); and 2.5% of awards (3% of total dollars) supported clinical trials. Public impact was measured by the annual average number of publications per grant (3.3±0.3) and cost ($154±24 thousand) per publication. Our analyses revealed that intussusceptive angiogenesis may represent an understudied mechanism, and therapeutic angiogenesis remains a remarkable challenge and opportunity to treat angiogenesis-related diseases. Angiogenesis denotes the process of new blood vessel formation and growth that is vital for normal embryonic development, patterning of the vascular system, and wound healing. Insufficient, excessive, or aberrant angiogenesis may have significant clinical consequences in diseases, such as stroke, myocardial infarction, cancer, chronic inflammation, and retinopathy.1 These key contributions to physiological and pathological processes support the high significance of research aimed at better understanding the complex regulation of angiogenesis. The National Institutes of Health (NIH), the largest funding source for biomedical research in the world, funds angiogenesis-related extramural research (hereafter abbreviated as …

Bladder Regeneration Using Multiple Acellular Scaffolds with Growth Factors in a Bladder.

Tissue engineering may become an alternative to current bladder augmentation techniques. Large scaffolds are needed for clinically significant augmentation, but can result in fibrosis and graft shrinkage. The purpose of this study was to investigate the use of multiple scaffolds instead of one large scaffold, to enhance bladder tissue regeneration and bladder capacity. Second, acellular collagen, collagen-heparin, and collagen-heparin scaffolds with growth factors (GFs) were used and the biological activity of the different scaffolds was compared in a large animal model. Scaffolds were made of bovine type I collagen with or without heparin (Ø = 3.2 cm). Collagen-heparin scaffolds were loaded with GFs, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and heparin-binding epidermal growth factor (HB-EGF). Three identical scaffolds prepared from collagen (COL-group), collagen with heparin (COLHEP-group), or collagen-heparin with growth factors (COLHEPGF-group) were implanted in one porcine bladder. The outcome was compared with sham-operated animals (Sham-group), in which no scaffold was used. Urodynamic evaluation was performed before surgery and 3 months after bladder reconstruction, together with histological evaluation. Survival rate was 92%, 12 animals completed the study, 3 of every group, 1 animal developed peritonitis due to urine leakage and was sacrificed. The regenerated area was largest in the COLHEP-group, and least in the COL-group (p = 0.002). Histological evaluation revealed a normal urothelial layer and good angiogenesis in all groups, and comparable ingrowth of smooth muscle cells. Urodynamics showed no statistically significant differences in bladder capacity and compliance between groups. Bladder capacity and compliance was very high in this animal model, which made it impossible to study the increase due to augmentation. Implantation of multiple collagen-heparin scaffolds in one bladder is feasible in a porcine model, resulting in tissue almost indistinguishable from native tissue involving all cell layers of the bladder. Collagen scaffolds with heparin incorporated resulted in a larger area of regenerated tissue. To reach clinically significant augmentation, multiple larger collagen-heparin scaffolds, with or without GFs, need to be tested to study the largest possible diameter of scaffold and number of used scaffolds still resulting in well-vascularized tissue.

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Isolevuglandins as a gauge of lipid peroxidation in human tumors

The cellular production of free radicals or reactive oxygen species (ROS) can lead to protein, lipid or DNA modifications and tumor formation. The cellular lipids undergo structural changes through the actions of enzymes (e.g. cyclooxygenases) or free radicals to form a class of compounds called Isolevuglandins (IsoLGs). The recruitment and continued exposure of tissue to ROS and IsoLGs causes increased cell proliferation, mutagenesis, loss of normal cell function and angiogenesis. The elevated concentration of ROS in cancerous tissues suggests that these mediators play an important role in cancer development. We hypothesized that tumors with elevated ROS levels would similarly possess an increased concentration of IsoLGs when compared with normal tissue. Using D11, an ScFv recombinant antibody specific for IsoLGs, we utilized immunohistochemistry to visualize the presence of IsoLG in human tumors compared to normal adjacent tissue (NAT) to the same tumor. We found that IsoLG concentrations were elevated in human breast, colon, kidney, liver, lung, pancreatic and tongue tumor cells when compared to NAT and believe that IsoLGs can be used as a gauge indicative of lipid peroxidation in tumors.

Normal Wound Healing and Tumor Angiogenesis as a Game of Competitive Inhibition.

Both normal wound healing and tumor angiogenesis are mitigated by the sequential, carefully orchestrated release of growth stimulators and inhibitors. These regulators are released from platelet clots formed at the sites of activated endothelium in a temporally and spatially controlled manner, and the order of their release depends on their affinity to glycosaminoglycans (GAG) such as heparan sulfate (HS) within the extracellular matrix, and platelet open canallicular system. The formation of vessel sprouts, triggered by angiogenesis regulating factors with lowest affinities for heparan sulfate (e.g. VEGF), is followed by vessel-stabilizing PDGF-B or bFGF with medium affinity for HS, and by inhibitors such as PF-4 and TSP-1 with the highest affinities for HS. The invasive wound-like edge of growing tumors has an overabundance of angiogenesis stimulators, and we propose that their abundance out-competes angiogenesis inhibitors, effectively preventing inhibition of angiogenesis and vessel maturation. We evaluate this hypothesis using an experimentally motivated agent-based model, and propose a general theoretical framework for understanding mechanistic similarities and differences between the processes of normal wound healing and pathological angiogenesis from the point of view of competitive inhibition.

Histone Deacetylase 5 Is Overexpressed in Scleroderma Endothelial Cells and Impairs Angiogenesis via Repression of Proangiogenic Factors

Vascular dysfunction represents a disease-initiating event in systemic sclerosis (SSc; scleroderma). Results of recent studies suggest that epigenetic dysregulation impairs normal angiogenesis and can result in abnormal patterns of blood vessel growth. Histone deacetylases (HDACs) control endothelial cell (EC) proliferation and regulate EC migration. Specifically, HDAC-5 appears to be antiangiogenic. This study was undertaken to test whether HDAC-5 contributes to impaired angiogenesis in SSc by repressing proangiogenic factors in ECs. Dermal ECs were isolated from patients with diffuse cutaneous SSc and healthy controls. Angiogenesis was assessed using an in vitro Matrigel tube formation assay. An assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed to assess and localize the genome-wide effects of HDAC5 knockdown on chromatin accessibility. The expression of HDAC5 was significantly increased in ECs from patients with SSc compared to healthy control ECs. Silencing of HDAC5 in SSc ECs restored normal angiogenesis. HDAC5 knockdown followed by ATAC-seq assay in SSc ECs identified key HDAC5-regulated genes involved in angiogenesis and fibrosis, such as CYR61, PVRL2, and FSTL1. Simultaneous knockdown of HDAC5 in conjunction with either CYR61, PVRL2, or FSTL1 inhibited angiogenesis in SSc ECs. Conversely, overexpression of these genes individually led to an increase in tube formation as assessed by Matrigel assay, suggesting that these genes play functional roles in the impairment of angiogenesis in SSc. Several novel HDAC5-regulated target genes associated with impaired angiogenesis were identified in SSc ECs by ATAC-seq. The results of this study provide a potential link between epigenetic regulation and impaired angiogenesis in SSc, and identify a novel mechanism for the dysregulated angiogenesis that characterizes this disease.

Time to Decide? Dynamical Analysis Predicts Partial Tip/Stalk Patterning States Arise during Angiogenesis.

Angiogenesis is a highly dynamic morphogenesis process; however, surprisingly little is known about the timing of the different molecular processes involved. Although the role of the VEGF-notch-DLL4 signaling pathway has been established as essential for tip/stalk cell competition during sprouting, the speed and dynamic properties of the underlying process at the individual cell level has not been fully elucidated. In this study, using mathematical modeling we investigate how specific, biologically meaningful, local conditions around and within an individual cell can influence their unique tip/stalk phenotype switching kinetics. To this end we constructed an ordinary differential equation model of VEGF-notch-DLL4 signaling in a system of two, coupled endothelial cells (EC). Our studies reveal that at any given point in an angiogenic vessel the time it takes a cell to decide to take on a tip or stalk phenotype may be drastically different, and this asynchrony of tip/stalk cell decisions along vessels itself acts to speed up later competitions. We unexpectedly uncover intermediate “partial” yet stable states lying between the tip and stalk cell fates, and identify that internal cellular factors, such as NAD-dependent deacetylase sirtuin-1 (Sirt1) and Lunatic fringe 1 (Lfng1), can specifically determine the length of time a cell spends in these newly identified partial tip/stalk states. Importantly, the model predicts that these partial EC states can arise during normal angiogenesis, in particular during cell rearrangement in sprouts, providing a novel two-stage mechanism for rapid adaptive behavior to the cells highly dynamic environment. Overall, this study demonstrates that different factors (both internal and external to EC) can be used to modulate the speed of tip/stalk decisions, opening up new opportunities and challenges for future biological experiments and therapeutic targeting to manipulate vascular network topology, and our basic understanding of developmental/pathological angiogenesis.

Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

Angiomotin like-1 is a novel component of the N-cadherin complex affecting endothelial/pericyte interaction in normal and tumor angiogenesis.

Transmission of mechanical force via cell junctions is an important component that molds cells into shapes consistent with proper organ function. Of particular interest are the cadherin transmembrane proteins, which play an essential role in connecting cell junctions to the intra-cellular cytoskeleton. Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving morphogenesis. We have previously identified the Amot protein family, which are scaffold proteins that integrate polarity, junctional, and cytoskeletal cues to modulate cellular shape in endothelial as well as epithelial cells. In this report, we show that AmotL1 is a novel partner of the N-cadherin protein complex. We studied the role of AmotL1 in normal retinal as well as tumor angiogenesis using inducible endothelial-specific knock-out mice. We show that AmotL1 is essential for normal establishment of vascular networks in the post-natal mouse retina as well as in a transgenic breast cancer model. The observed phenotypes were consistent with a non-autonomous pericyte defect. We show that AmotL1 forms a complex with N-cadherin present on both endothelial cells and pericytes. We propose that AmotL1 is an essential effector of the N-cadherin mediated endothelial/pericyte junctional complex.

Engineered mesenchymal cell-based patches as controlled VEGF delivery systems to induce extrinsic angiogenesis

Therapeutic over-expression of Vascular Endothelial Growth Factor (VEGF) by transduced progenitors is a promising strategy to efficiently induce angiogenesis in ischemic tissues (e.g. limb muscle and myocardium), but tight control over the micro-environmental distribution of the dose is required to avoid induction of angioma-like tumors. Therapeutic VEGF release was achieved by purified transduced adipose mesenchymal stromal cells (ASC) that homogeneously produce specific VEGF levels, inducing only normal angiogenesis after injection in non-ischemic tissues. However, the therapeutic potential of this approach mostly in the cardiac field is limited by the poor cell survival and the restricted area of effect confined to the cell-injection site. The implantation of cells previously organized in vitro in 3D engineered tissues could overcome these issues. Here we hypothesized that collagen sponge-based construct (patch), generated by ASC expressing controlled VEGF levels, can function as delivery device to induce angiogenesis in surrounding areas (extrinsic vascularization). A 7-mm-thick acellular collagen scaffold (empty), sutured beneath the patch, provided a controlled and reproducible model to clearly investigate the ongoing angiogenesis in subcutaneous mice pockets. VEGF-expressing ASC significantly increased the capillary in-growth inside both the patch itself and the empty scaffold compared to naïve cells, leading to significantly improved survival of implanted cells.These data suggest that this strategy confers control (i) on angiogenesis efficacy and safety by means of ASC expressing therapeutic VEGF levels and (ii) over the treated area through the specific localization in an engineered collagen sponge-based patch. Statement of SignificanceDevelopment of efficient pro-angiogenic therapies to restore the micro-vascularization in ischemic tissues is still an open issue. Although extensively investigated, the promising approach based on injections of progenitors transduced to over-express Vascular Endothelial Growth Factor (VEGF) has still several limitations: (i) need of a tight control over the microenvironmental VEGF dose to avoid angioma-like tumor growth; (ii) poor implanted cell survival; (iii) effect area restricted mainly to the injection sites. Here, we aimed to overcome these drawbacks by generating a novel cell-based controlled VEGF delivery device. In particular, transduced mesenchymal cells, purified to release a sustained, safe and efficient VEGF dose, were organized in three-dimensional engineered tissues to improve cell survival and provide a uniform vascularization throughout both the mm-thick implanted constructs themselves and the surrounding area.

Abstract 1203: Targeting activated tumor vasculature with human Granzyme B variants engineered for improved stability and activity

Abstract Angiogenesis is a critical process in numerous diseases, and targeting tumor neovasculature has therapeutic value in controlling tumor progression and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) and its receptors VEGFR-1 and VEGFR-2 have been implicated as central mediators of normal angiogenesis and tumor neovascularization. VEGF121 is a naturally-occurring VEGF-A splice variant that binds to these receptors, which are over-expressed on the endothelium of activated tumor vasculature or neovasculature but not normal vasculature. Granzyme B (GrB) plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade through both caspase-dependent and -independent mechanisms. The fusion protein GrB/VEGF121 expressed in mammalian cells and purified to homogeniety was lethal to tumor and endothelial cells in vitro in a manner that correlated closely to total VEGFR-2 expression. IC50 levels were found to be in the nanomolar range. GrB/VEGF121 internalized rapidly into VEGFR-2 expressing cells while the internalization into VEGFR-1 expressing cells was significantly reduced. We engineered GrB variants for improved protein production and for resistance to serpin B9 (PI-9), a natural inhibitor of Granzyme B found at varying levels in cells and plasma, and compared these GrB/VEGF121 constructs directly to unmodified GrB/VEGF121. Variants took into account the impact of the mutation on substrate specificity, and the ability of the mutated GrB to cleave its various cellular substrates. As expected, mutation of the active site 195serine to alanine (S195A) resulted in a complete loss of GrB enzymatic activity as well as in vitro cytotoxicity against VEGFR-1+ and VEGFR-2+ cells. Enzymatic activity in PBS of the K27E, R28A (“EA-GV”) double mutant was identical to that of GrB/VEGF121. In 50% human serum, however, the enzymatic activity of GrB/VEGF121 steadily declined to less than 10% over 24 hours while the enzymatic activity of EA-GV remained over 40% during that time period. This construct retained higher cytotoxic activity after pre-incubation in serum for 4h, compared to GrB/VEGF121. C-GrB/VEGF121, without an unpaired cysteine, resulted in reduced aggregation and a vastly improved yield with otherwise similar serum stability and in vitro cytotoxicity as unmodified GrB/VEGF121. The pharmacokinetic profile for C-GrB/VEGF121 indicated a t1/2α of 6.6 min and t1/2β of 33.3 h. A limited profile for EA-GV indicates a longer terminal-phase half-life for this construct compared to C-GrB/VEGF121. Our studies suggest that these novel GrB variants may significantly improve in vivo half-life. Further studies are underway to determine whether these modifications impact production of soluble material and in vivo therapeutic efficacy against established tumor xenograft models. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. Targeting activated tumor vasculature with human Granzyme B variants engineered for improved stability and activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1203.

Integrin β8 Deletion Enhances Vascular Dysplasia and Hemorrhage in the Brain of Adult Alk1 Heterozygous Mice.

Brain arteriovenous malformation (bAVM), characterized by tangled dysplastic vessels, is an important cause of intracranial hemorrhage in young adults, and its pathogenesis and progression are not fully understood. Patients with haploinsufficiency of transforming growth factor-β (TGF-β) receptors, activin receptor-like kinase 1 (ALK1) or endoglin (ENG) have a higher incidence of bAVM than the general population. However, bAVM does not develop effectively in mice with the same haploinsufficiency. The expression of integrin β8 subunit (ITGB8), another member in the TGF-β superfamily, is reduced in sporadic human bAVM. Brain angiogenic stimulation results at the capillary level of vascular malformation in adult Alk1 haploinsufficient (Alk1 +/- ) mice. We hypothesized that deletion of Itgb8 enhances bAVM development in adult Alk1 +/- mice. An adenoviral vector expressing Cre recombinase (Ad-Cre) was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brain of Alk1 +/-;Itgb8-floxed mice to induce focal Itgb8 gene deletion and angiogenesis. We showed that compared with Alk +/- mice (4.75±1.38/mm2), the Alk1 +/-;Itgb8-deficient mice had more dysplastic vessels in the angiogenic foci (7.14±0.68/mm2, P=0.003). More severe hemorrhage was associated with dysplastic vessels in the brain of Itgb8-deleted Alk1 +/- , as evidenced by larger Prussian blue-positive areas (1278±373pixels/mm2 vs. Alk1 +/- :320±104pixels/mm2; P=0.028). These data indicate that both Itgb8 and Alk1 are important in maintaining normal cerebral angiogenesis in response to VEGF. Itgb8 deficiency enhances the formation of dysplastic vessels and hemorrhage in Alk1 +/- mice.

CD34 Promotes Pathological Epi-Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy.

The sialomucins CD34 and podocalyxin (PODXL) are anti-adhesive molecules expressed at the luminal membrane of endothelial cells of small blood vessels and facilitate vascular lumen formation in the developing mouse aorta. CD34 transcript and protein levels are increased during human angiogenesis, its expression is particularly enriched on endothelial tip cell filopodia and CD34 is a marker for tip cells in vitro. Here, we investigated whether CD34 merely marks endothelial tip cells or has a functional role in tip cells and angiogenesis. We assessed that silencing CD34 in human microvascular endothelial cells has little effect on endothelial cell migration or invasion, but has a significant effect on vascular-endothelial growth factor-induced angiogenic sprouting activity in vitro. In vivo, the absence of CD34 reduced the density of filopodia on retinal endothelial tip cells in neonatal mice, but did not influence the overall architecture of the retinal vascular network. In oxygen-induced retinopathy, Cd34-/- mice showed normal intra-retinal regenerative angiogenesis but the number of pathological epi-retinal neovascular tufts were reduced. We conclude that CD34 is not essential for developmental vascularization in the retina, but its expression promotes the formation of pathological, invasive vessels during neovascularization.

Endothelial cell transplantation in tumors restores normal vasculature, reduces tumor hypoxia, and suppresses tumor outgrowth

ObjectivesVascular normalization, or restoration of the normal structure and function of blood vessels, using molecular-targeted therapy, has emerged as a potential strategy for treating malignant cancer and other vascular disorders. We hypothesized that restoring tumor blood vessels to their normal state would alleviate hypoxic conditions and potentially enhance the delivery of anticancer drugs. Our objective was to determine if transplanting normal endothelial cells into tumor-bearing mice could trigger vascular normalization. MethodsTumor cells were injected into the dorsal subcutis of severe combined immunodeficiency (SCID) mice (day 0). Tumor-bearing mice were injected intraperitoneally with cisplatin at day 14 to create scaffolds for blood vessel formation in the tumors. At day 28, human microvascular endothelial cells (HMVECs) or human embryonic stem-derived endothelial cells (ESECs) were transplanted into the necrotic regions of the tumor to induce normal angiogenesis. ResultsMicroscopic observation revealed that the transplanted HMVECs or ESECs formed anastomoses with the host mouse vasculature. In addition, blood vessels with blood flow could be detected after 14d. Blood vessels reconstituted by HMVECs or ESECs exhibited normal vasculature, and tumor growth was significantly inhibited upon treatment. ConclusionReconstruction of tumor blood vessels to their normal state alleviated hypoxic conditions and improved the efficiency of drug delivery; the present approach provides a useful model for the development of new cancer therapies.

Total oxidative and anti-oxidative status, and ADAMTS-12 levels in placenta previa and early-onset severe preeclampsia.

This study was carried out due to the discussions in the literature stating that the inverse association between placenta previa (PP) and preeclampsia (PE). The aim of this study was to determine whether total antioxidant status (TAS), and total oxidant status (TOS) and ADAMTS-12 levels differ among early-onset (<34th gestational week) severe PE (EOS-PE), PP and uncomplicated pregnancies. In this case-control study, serum samples obtained from 26 pregnant with EOS-PE, 31 pregnant with PP, and 32 healthy patients with uncomplicated pregnancies (control group). TOS levels were significantly higher in the EOS-PE than in the control group and PP groups (p=0.002, p=0.05, respectively). TAS levels were significantly lower in the EOS-PE than in the control (p<0.001). Although TAS levels were lower in the EOS-PE group than in the PP group, the differences were not statistically significant (p=0.09). There were no significant differences in the ADAMTS-12 levels of the groups. The data in this study suggested that the balance between oxidative and anti-oxidative substances were comparable and normal in pregnancies complicated by PP when compared to normal pregnancies without placentation abnormality. In support of this, we encountered no case with PE and fetal growth restriction in our study groups suggesting normal placental angiogenesis. Contrarily, EOS-PE was associated with decreased TAS and increased TOS levels in the maternal serum.

Integrated analysis of mRNA and miRNA to unravel novel mechanisms of sunitinib long term response in mRCC.

e16080Background: Normal and pathological angiogenesis is under the control of various developmental genetic programs, including Notch and Hedgehog pathways. More recently, miRNAs have emerged as k...

Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF164 in skeletal muscle.

Therapeutic angiogenesis by growth factor delivery is an attractive treatment strategy for ischemic diseases, yet clinical efficacy has been elusive. The angiogenic master regulator VEGF-A can induce aberrant angiogenesis if expressed above a threshold level. Since VEGF remains localized in the matrix around expressing cells, homogeneous dose distribution in target tissues is required, which is challenging. We found that co-expression of the pericyte-recruiting factor PDGF-BB at a fixed ratio with VEGF from a single bicistronic vector ensured normal angiogenesis despite heterogeneous high VEGF levels. Taking advantage of a highly controlled gene delivery platform, based on monoclonal populations of transduced myoblasts, in which every cell stably produces the same amount of each factor, here we rigorously investigated a) the dose-dependent effects, and b) the long-term safety and stability of VEGF and PDGF-BB co-expression in skeletal muscle. PDGF-BB co-expression did not affect the normal angiogenesis by low and medium VEGF doses, but specifically prevented vascular tumors by high VEGF, yielding instead normal and mature capillary networks, accompanied by robust arteriole formation. Induced angiogenesis persisted unchanged up to 4 months, while no tumors appeared. Therefore, PDGF-BB co-expression is an attractive strategy to improve safety and efficacy of therapeutic angiogenesis by VEGF gene delivery.

The Actin Cytoskeleton Mediates Hyperoxia Response of Patient-Derived Primary Endothelial Progenitor Cells through Thymosin BETA-4 and Hypoxia-Inducible Factor Signaling

Disruptions to pro-growth, proliferation, and angiogenesis signaling pathways within endothelial progenitor cells (EPC) may play key role in the pathogenesis of multiple pediatric pulmonary diseases, such as bronchopulmonary dysplasia. Using high-throughput, single-molecule, single-cell imaging of multiple RNA and protein species, we have created spatiotemporal “stop-motion” movies of F-actin structure, thymosin beta-4 (Tβ4), hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) expression in a unique resource of primary, patient-derived EPC isolated from the umbilical cord blood of preterm- and term-birth infants, grown in vitro using differing oxygen environments. In particular, a hyperoxic growth environment mimics preterm birth, raising the environmental oxygen level at an abnormal time point during fetal development. We find that EPC isolated from preterm- versus term-birth infants have different spatial expression of Tβ4 and HIF-1α protein at basal conditions and that upon exposure to hyperoxia, the two classes of EPC respond with markedly different spatiotemporal expression of Tβ4/HIF-1α protein and VEGF/eNOS RNA. Additionally, hyperoxia causes a remodeling of the actin cytoskeleton that releases excess Tβ4, an actin sequestering protein, into the cytosol. Because preterm EPC have an elevated cytosolic level of Tβ4 at basal conditions compared to term EPC, this excess of Tβ4 activates an inhibitory feedback network to reduce Tβ4 levels and subsequently limits the translocation of HIF-1α, a potent transcription factor for the VEGF-eNOS signaling pathway, to the nucleus. Overall these signaling changes due to hyperoxia have the effect of down-regulating growth, proliferation, and angiogenesis in EPC isolated from preterm-birth infants. Our results suggest a new potential pathway for molecular intervention that may restore normal angiogenesis after preterm birth.

Aminoacyl-Transfer RNA Synthetase Deficiency Promotes Angiogenesis via the Unfolded Protein Response Pathway.

Understanding the mechanisms regulating normal and pathological angiogenesis is of great scientific and clinical interest. In this report, we show that mutations in 2 different aminoacyl-transfer RNA synthetases, threonyl tRNA synthetase (tars(y58)) or isoleucyl tRNA synthetase (iars(y68)), lead to similar increased branching angiogenesis in developing zebrafish. The unfolded protein response pathway is activated by aminoacyl-transfer RNA synthetase deficiencies, and we show that unfolded protein response genes atf4, atf6, and xbp1, as well as the key proangiogenic ligand vascular endothelial growth factor (vegfaa), are all upregulated in tars(y58) and iars(y68) mutants. Finally, we show that the protein kinase RNA-like endoplasmic reticulum kinase-activating transcription factor 4 arm of the unfolded protein response pathway is necessary for both the elevated vegfaa levels and increased angiogenesis observed in tars(y58) mutants. Our results suggest that endoplasmic reticulum stress acts as a proangiogenic signal via unfolded protein response pathway-dependent upregulation of vegfaa.