Gene Expression Of Angiogenic Factors Research Articles

Exogenous melatonin ameliorates embryo-maternal cross-talk in early pregnancy in sheep.

Early pregnancy losses cause 25% of pregnancy failures in small ruminants because of an asynchrony between conceptus and uterine signals. In that context, melatonin plays a crucial role in sheep reproductive dynamics, but little is known about its effects in the peri-implantation period. We hypothesized that melatonin supports embryo implantation by modulating the uterine microenvironment. This study aimed to assess the effects of exogenous melatonin on the endometrium and early placenta rearrangement. Ten multiparous ewes either did (MEL, n=5) or did not (CTR, n=5) receive a subcutaneous melatonin implant (18 mg) 50 d before a synchronized mating. On Day 21 of pregnancy, sheep were euthanized. MEL ewes exhibited a higher prolificity rate (2.8 vs. 2.0 embryos/ewe) and plasma progesterone levels (3.84 vs. 2.96 ng/mL, P<0.05) than did CTR ewes. Groups did not differ significantly in embryo crown-rump length. MEL placenta had significantly (P<0.001) more binucleated trophoblast cells in the chorion region and ovine placental lactogen expression was significantly (P<0.05) more strongly upregulated than it was in CTR. Exogenous melatonin increased significantly (P<0.05) gene expression of angiogenic factors (VEGFA, VEGFR1, IGF1R), IFNAR2, and PR in the caruncular endometrium. Expression of the MT2 receptor in the endometrium and placenta was significantly (P<0.05) higher in MEL group. Results indicate that melatonin implants acted differentially on the uterus and placenta rearrangement. Melatonin increases differentiation in the placenta and induces changes that could promote vessel maturation in the endometrium, which suggests that it enhances the uterine microenvironment in the early stage of pregnancy in sheep.

283 Awardee Talk: Effects of Late Gestational Maternal Nutrient Balance on Nutrient Partitioning to the Gravid Uterus in Beef Cattle

Abstract Successful intrauterine growth relies on adequate placental nutrient transfer capacity and is determined by placental size, placental morphology, uteroplacental blood supply, nutrient transporter abundance, and the nutrients available in maternal circulation. The majority of bovine fetal growth occurs during late gestation and must be accompanied by similar exponential increases in uteroplacental development to allow for necessary nutrient delivery. Competition for nutrients in young females between maternal growth and satisfying pregnancy, lactation, and maintenance requirements is still poorly understood. Our current work investigated the relationships between maternal, fetal, and uteroplacental factors and the impacts of late gestational nutrient restriction on prenatal nutrient delivery to the calf. Analysis of 113 full-term expelled placentas revealed that primiparous dams and dams in thin body condition had smaller placentas than their older or fleshier counterparts, suggesting the addition of growth requirements or exhausted nutrient reserves limited placental and fetal development. Calf size at birth and placental weight were positively correlated among all females, but it is still unknown which controls and signals for the growth of the other. Uterine blood flow measured during late gestation was positively correlated with calf birth weight; however, uterine blood flow and placental weight were only positively correlated closer to parturition at 40 and 20 d before calving. Additionally, contralateral uterine blood flow may have a disproportionately greater contribution to placental and fetal growth as it more closely resembled total blood flow associations than did ipsilateral. In 2 experiments, primiparous females fed 70% of energy and protein requirements during late gestation lost body weight and condition, had less metabolites in circulation, and had lower maternal heart rates than those fed to meet requirements. In Exp. 1, nutrient restricted dams had less contralateral placental growth, yet total uterine blood flow and placental weight were maintained and ensured fetal growth similar to control offspring. In Exp. 2, nutrient restriction reduced calf birth weight by 15%. Fetal growth perturbations were largely driven by a 17% reduction in placental size along with less nutrients available from maternal circulation, independent of a concomitant decrease in uterine blood flow. At 48 h of age, calf visceral organs were smaller due to nutrient restriction, but asymmetrical growth allowed for growth of the brain, lungs, and bone to be spared. Ongoing analysis of cotyledonary nutrient transporter and angiogenic factor gene expression will provide crucial insight into another determinant of placental nutrient transfer capacity. These findings demonstrate that pregnant beef females in a negative nutrient balance will attempt to spare fetal growth but at a certain threshold placental adaptation can be compromised, resulting in intrauterine growth restriction. A better understanding of nutrient delivery to the gravid uterus will allow for improved management strategies to ensure optimal fetal growth.

Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue

BackgroundOvarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex.MethodsCryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24–36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150–200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks.ResultsA significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045).ConclusionsNeedle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.

PSI-2 Disruption of CXCL12-CXCR4 Axis at Fetal-Maternal Interface During Implantation Alters Ovine Angiogenic Expression at Mid-to-Late Gestation

Abstract Placental dysfunction originates during placental development, specifically the events of placentation. Vascularization is a key component of placentation due to its role in fetal-maternal exchange. Cytokines are regulators of placental development; signaling of chemokine ligand 12 (CXCL12) and its receptor (CXCR4) drives placental vascularization. Utilizing an in vivo sheep model, we demonstrated that suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface reduces placental vascularization. We hypothesized these negative impacts early in gestation would result in compromised placental development and vascularization at mid to late gestation. On day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes and delivered either CXCR4 inhibitor (AMD3100) at 1x dose (n = 8),1.5X dose (n = 8), or 3X dose (n = 8) or saline (n = 13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On day 90 and day 135 placental tissues were collected and maternal (caruncle) and fetal (cotyledon) placenta components were separated and analyzed. Gene expression of angiogenic factors were analyzed using Real-time qPCR. Gene expression was significantly altered by AMD3100 treatment for the vascular endothelial growth factor receptors (FLT-1 and KDR) primarily in day 90 placenta contralateral to the corpus luteum. Significant decreases in KDR were seen in 3x compared to control (P &amp;lt; 0.03), 3x compared to 1.5x (P &amp;lt; 0.008), and 3x contralateral compared to ipsilateral (P &amp;lt; 0.01); however, FLT-1 significantly increased (P &amp;lt; 0.02) from control to 1.5x. Day 90 caruncle tissue had significant increases in FLT-1 from control to 3x (0.02), 1.5x to 3x (P &amp;lt; 0.03); however, 1.5x treatment FLT-1 significantly decreased (P &amp;lt; 0.02) ipsilateral to contralateral. Fibroblast growth factor 2 increased in both contralateral 3x compared to control cotyledon (P &amp;lt; 0.03) and caruncle (P &amp;lt; 0.03). Our data underscore the importance of CXCL12-CXCR4 signaling during placentation and provide strong evidence that altering CXCR4-mediated signaling during early placentation induces enduring effects altering vascular development later in gestation. A greater understanding of CXCL12/CXCR4 functions may reveal methods to improve reproductive success and fetal health.

Enzymatic self-assembly nanofibers anchoring mesenchymal stem cells induce cell spheroids and amplify paracrine function for myocardial infarction therapy

Transplantation of stem cells with biomaterials has proved to be an effective way to improve cell engraftment and survival in treating degenerative diseases. Owing to their structural resemblance to the extracellular matrix (ECM), synthetic biomaterials have been shown to mimic the native tissue microenvironment and support regeneration. Thus, they have been used extensively in tissue engineering to induce desirable cellular responses. However, it is challenging to develop synthetic materials for mimicking native ECM with dynamic processes like cell-mediated remodeling. Here, inspired by the natural unfolding of fibronectin to dynamic ECM remodeling, we designed an enzyme-instructed self-assembled peptide (Nap-pD-E7) that enables a sheet of mesenchymal stem cells (MSC) to form cell spheroids during the intercellular morphological transition of enzymatic self-assembly. Analysis showed that the supramolecular assemblies formed by phosphatase instructed partial dephosphorylation of Nap-pD-E7 interacted with ECM molecules in the intercellular space of MSC. Further studies revealed that Nap-pD-E7 assemblies significantly improved the gene expression of angiogenic factors and that conditioned medium from Nap-pD-E7–induced MSC spheroids promoted tube formation in human umbilical vein endothelial cells (HUVECs). Finally, transplantation of Nap-pD-E7 hydrogel encapsulated MSC spheroids to the murine infarcted myocardium could improve cell retention, promote tissue angiogenesis, attenuate ventricular remodeling, therefore ameliorate heart function. In conclusion, this approach using cell-responsive peptide assemblies mimicking cell-mediated remodeling of the ECM to modulate MSC functions is a feasible option for cell-based therapy.

Angiogenesis during coronal pulp regeneration using rat dental pulp cells: Neovascularization in rat molars in vivo and proangiogenic dental pulp cell-endothelial cell interactions in vitro

Background/purposeAngiogenesis is considered a crucial event for dental pulp regeneration. The purpose of this study was to demonstrate neovascularization during coronal pulp regeneration in rat molars using rat dental pulp cells (rDPCs) and to examine whether rDPC-endothelial cell interactions promote proangiogenic capacity in vitro. Materials and methodsMaxillary first molars of Wistar rats (n = 42) were pulpotomized and rDPCs isolated from incisors were implanted with a porous poly (l-lactic acid) (PLLA) scaffold and hydrogel (Matrigel). After 3, 7, and 14 days, coronal pulp tissues were examined histologically and by nestin and CD146 immunohistochemistry. rDPCs and rat dermal microvascular endothelial cells (rDMECs) were cocultured for 4 days and vascular endothelial growth factor (VEGF) synthesis and angiogenic factor gene expression were determined by enzyme-linked immunosorbent assays and real-time polymerase chain reaction, respectively. Effects of cocultured medium on tube formation by rDMECs were also evaluated. ResultsImplantation of rDPC/PLLA/Matrigel induced coronal pulp regeneration with dentin bridge formation and arrangement of nestin-positive odontoblast-like cells at 14 days. PLLA/Matrigel without rDPCs did not induce pulp regeneration. CD146-positive blood vessels increased in density in the remaining pulp tissues at 3 and 7 days, and in the regenerated pulp tissue at 14 days. rDPC/DMEC coculture significantly promoted VEGF secretion and mRNA expression of nuclear factor-kappa B, angiogenic chemokine CXCL1, and chemokine receptor CXCR1. Cocultured medium significantly promoted tube formation. ConclusionCoronal pulp regeneration with rDPC/PLLA/Matrigel was accompanied by neovascularization. rDPC-rDMEC interactions may promote angiogenic activity represented by proangiogenic factor upregulation and tube formation in vitro.

Angiogenic Potential of VEGF Mimetic Peptides for the Biofunctionalization of Collagen/Hydroxyapatite Composites.

Currently, the focus on bioinspired concepts for the development of tissue engineering constructs is increasing. For this purpose, the combination of collagen (Coll) and hydroxyapatite (HA) comes closest to the natural composition of the bone. In order to confer angiogenic properties to the scaffold material, vascular endothelial growth factor (VEGF) is frequently used. In the present study, we used a VEGF mimetic peptide (QK) and a modified QK-peptide with a poly-glutamic acid tag (E7-QK) to enhance binding to HA, and analyzed in detail binding efficiency and angiogenic properties. We detected a significantly higher binding efficiency of E7-QK peptides to hydroxyapatite particles compared to the unmodified QK-peptide. Tube formation assays revealed similar angiogenic functions of E7-QK peptide (1µM) as induced by the entire VEGF protein. Analyses of gene expression of angiogenic factors and their receptors (FLT-1, KDR, HGF, MET, IL-8, HIF-1α, MMP-1, IGFBP-1, IGFBP-2, VCAM-1, and ANGPT-1) showed higher expression levels in HUVECs cultured in the presence of 1 µM E7-QK and VEGF compared to those detected in the negative control group without any angiogenic stimuli. In contrast, the expression of the anti-angiogenic gene TIMP-1 showed lower mRNA levels in HUVECs cultured with E7-QK and VEGF. Sprouting assays with HUVEC spheroids within Coll/HA/E7-QK scaffolds showed significantly longer sprouts compared to those induced within Coll/HA/QK or Coll/HA scaffolds. Our results demonstrate a significantly better functionality of the E7-QK peptide, electrostatically bound to hydroxyapatite particles compared to that of unmodified QK peptide. We conclude that the used E7-QK peptide represents an excellently suited biomolecule for the generation of collagen/hydroxyapatite composites with angiogenic properties.

Effects of bioactive glass on proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells

OBJECTIVE To investigate the effects of phytic acid derived bioactive P2O5-SiO2-CaO gel-glasses (PSC) on the proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. METHODS HUVECs were cultured in PSC extracts, which were prepared with endothelial cell medium (ECM) at a gradient concentration of 0.01, 0.1, 1 and 2 g/L. Cells cultured in ECM were used as the control. The effect of PSC on HUVECs proliferation was assessed on the 1st, 3rd, 5th, 7th and 10th days with (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide assay (MTT), and the optimum PSC concentration for HUVECs proliferation was used in the following experiments. The subsequent experiments were divided into two groups. The experimental group used PSC extracts to culture HUVECs (PSC group) and the control group used ECM to culture HUVECs (ECM group). Gene expression of angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), was detected on the 2nd, 4th and 7th days by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). The morphology and number of tubules formation were observed at the 4th and 10th hours. Image J software was used for counting and quantitative analysis. RESULTS The results of MTT assay showed that 0.1 g/L PSC group had the most significant effect on promoting HUVECs proliferation. The optical density values of 0.1 g/L PSC group on the 5th and 7th days were significantly higher than those of the other PSC groups and the control group (P < 0.05). The result of real-time RT-PCR showed that 0.1 g/L PSC extract up-regulated the mRNA expression of VEGF and bFGF significantly (P < 0.05). On the 4th day, the gene expressions of VEGF and bFGF in PSC group were 1.59 and 1.45 times higher than those in ECM group respectively, and on the 7th day, the gene levels of VEGF and bFGF in PSC group were 1.98 and 1.37 times higher than those in ECM group respectively. The tubule formation assay showed that the maturity and density of the tubules in 0.1 g/L PSC group was much better than that in the ECM group at the 10th hour. The quantitative analysis by Image J indicated that the tubules number in PSC group (29.63±2.29) was higher than in the ECM group (20.13±2.36), with statistical significance (P < 0.05). CONCLUSION PSC showed significant promoting effects on HUVECs' proliferation, differentiation and angiogenesis in vitro.

Abstract 13681: Single-cell Gene Profiling Revealed Heterogeneity of Human Skeletal Muscle Derived Stem Cells and Their Paracrine Factors

Background: Transplantation of sheet-shaped autologous skeletal muscle-derived cells (SMDCs) has been shown to produce therapeutic effects via paracrine factors in ischemic cardiomyopathy. SMDCs are heterogeneous cell population consist of myoblasts and non-myogenic cells, and their detailed characteristic and paracrine factors has not been fully analyzed. We herein applied single-cell RNA sequencing (scRNA-seq) to profile the population and paracrine factors of SMDCs. Methods and Results: Primary SMDCs of human origin were subjected to scRNA-seq and divided into 5 populations, expressing myogenic markers differently. These included four myogenic populations with higher expression of MYF5 (myoblasts), myogenin (early differentiated cells), mitosis-related gene (proliferating cells), and MYL3 (moderately differentiated cells) and non-myogenic population with lower expression of myogenic genes. On the other hand, PAX7 , quiescent satellite cell marker, and MYH4 , terminally differentiated marker, were not expressed in any populations. Furthermore, regarding the expression of key angiogenic factors, VEGF and HGF were highly expressed in the non-myogenic population and HMGB1 was expressed in both populations. Moreover, analysis of the gene expression of angiogenic factors differentially expressed in each population indicated that myoblast population highly expressed PDGFA and CCN (Cellular communication network factor) 1 , and early differentiated population highly expressed CCN2 . Although these factors were expressed in other populations, their expression tended to be lower in the moderately differentiated cell population. Conclusion: Analysis of SMDCs using scRNA-seq revealed that they are heterogeneous cells consisting of multiple myogenic populations with different degrees of differentiation and cytokine production, suggesting that single-cell gene profiling may be useful for the evaluation on characters of effector cells.

1683-P: Upregulation of Pancreatic Islet EGF Receptor Improves Beta-Cell Identity and In Vivo Vascularisation in a Directly Observed Transplant Model

Aims: Long-term outcomes for islet transplantation remain suboptimal. An important determinant of islet transplantation success is vascularisation of engrafted islets. The epidermal growth factor receptor (EGFR) is a key angiogenic and pro-survival factor. In an in vivo mouse model, we investigated if EGFR overexpression in pancreatic islets improves engraftment. Methods: Donor islets from C57BL/6 mice were infected with adenoviruses generated to encoding human EGFR alongside a GFP reporter. To assess the effects of EGFR over-expression on beta-cell identity and function, qPCR was run to determine expression of angiogenesis, beta-cell enriched and beta-cell disallowed genes compared with islets infected with control (empty vector) virus. To assess the effects of EGFR-overexpression on implantation in vivo, EGFR-overexpressing or control (empty vector) islets were transplanted into the anterior chambers of the eyes of syngeneic recipients. Using confocal microscopy, islet volume (GFP signal) and blood vessel density (following IV administration of dextran conjugated Texas Red) were assessed regularly and longitudinally over 30 days. Results: qPCR results showed that EGFR-overexpressing islets had greater gene expression of angiogenic factors. Consistent with preserved beta-cell identity, beta-cell enriched genes were upregulated in EGFR-overexpressing islets compared to control islets, whilst beta-cell disallowed genes were repressed. EGFR-overexpressing islets demonstrating greater density compared to control islets at all time points (n=5 animals per group, 1-5 islets per eye, p&amp;lt;0.05). Conclusion: We are the first to demonstrate that EGFR overexpression can promote the vascularisation of transplanted islets in vivo. The transcriptional profiling of these islets suggests an improved potential for beta-cell regenerative capacity and function. Disclosure V. Salem: None. U. Ali: None. K. Suba: None. S. Bitsi: None. T. Lopes: None. A. Martin Alonso: None. Y.S. Patel: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. B. Owen: None. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. S.M. Rothery: None. A. Tomas: None. Funding Diabetes UK (15/0005317)

Expression of angiogenic factors in the uteroplacental unit is altered at time of placentation in a porcine model of von Willebrand disease type 1

Von Willebrand disease (VWD) affects blood coagulation and correlates with angiodysplasia. Data on VWD-affected women point to slightly increased miscarriage rates. We aimed to investigate the impact of VWD on angiogenesis in the uteroplacental unit of pregnant pigs of a model of VWD type 1 (T1). Uteri, placentae, and embryos were harvested at time of placentation (day 29 to 31) from four sows (two wildtype (WT) and two heterozygous for a von Willebrand factor (VWF) mutation diagnosed with T1). T1 sows were bred to a T1 boar creating embryos of three different genotypes: WT, T1 or homozygous for the VWF mutation corresponding with VWD type 3 (T3). Uteroplacental tissues were examined histologically. Embryos were genotyped. Gene expression of angiogenic factors possibly related to VWF was determined by quantitative real-time PCR. Corresponding protein expression was analyzed by immunohistochemistry. Genotyping revealed 35.3% WT, 52.9% T1 and 5.9% T3 embryos (5.9% not classified confidently). No histological alterations were found. Gene expression of VEGF was significantly increased in T1 placentae while expression of ANG1, ANG2, TIE2, and ITGB3 was significantly reduced, confirmed on protein level for different cell types. TIE2/TIE1 ratios were significantly lower in T1 placentae. Distribution of embryo genotypes indicates selection favoring the WT. Significant expression differences of angiogenic factors in placentae suggest influence of VWF on these factors during placentation, although angiodysplasia was not observed. The alterations concerning VEGF/VEGFR-2 signaling, integrin expression and the ANG/TIE system may influence angiogenesis and vascular adaptation during placentation and thus the overall outcome of pregnancy.

The acute angiogenic signalling response to low-load resistance exercise with blood flow restriction

This study investigated protein kinase activation and gene expression of angiogenic factors in response to low-load resistance exercise with or without blood flow restriction (BFR). In a repeated measures cross-over design, six males performed four sets of bilateral knee extension exercise at 20% 1RM (reps per set = 30:15:15:continued to fatigue) with BFR (110 mmHg) and without (CON). Muscle biopsies were obtained from the vastus lateralis before, 2 and 4 h post-exercise. mRNA expression was determined using real-time RT–PCR. Protein phosphorylation/expression was determined using Western blot. p38MAPK phosphorylation was greater (p = 0.05) at 2 h following BFR (1.3 ± 0.8) compared to CON (0.4 ± 0.3). AMPK phosphorylation remained unchanged. PGC-1α mRNA expression increased at 2 h (5.9 ± 1.3 vs. 2.1 ± 0.8; p = 0.03) and 4 h (3.2 ± 0.8 vs. 1.5 ± 0.4; p = 0.03) following BFR exercise with no change in CON. PGC-1α protein expression did not change following either exercise. BFR exercise enhanced mRNA expression of vascular endothelial growth factor (VEGF) at 2 h (5.2 ± 2.8 vs 1.7 ± 1.1; p = .02) and 4 h (6.8 ± 4.9 vs. 2.5 ± 2.7; p = .01) compared to CON. mRNA expression of VEGF-R2 and hypoxia-inducible factor 1α increased following BFR exercise but only eNOS were enhanced relative to CON. Matrix metalloproteinase-9 mRNA expression was not altered in response to either exercise. Acute low-load resistance exercise with BFR provides a targeted angiogenic response potentially mediated through enhanced ischaemic and shear stress stimuli.

Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing.

ObjectivesThe objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing.MethodsHuman PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically.ResultsThe total number of PBMNCs was decreased after QQ-culture, however, the number of CD34+ and CD206+ cells were found to have increased as assessed by flow cytometry analysis. In addition, gene expression of angiogenic factors was upregulated in QQMNCs. In the animal model, the rate of bone union was higher in the QQMNC group than in the other groups. Radiographic scores and bone volume were significantly associated with the enhancement of angiogenesis in the QQMNC group.ConclusionWe have demonstrated that QQMNCs have superior potential to accelerate fracture healing compared with PBMNCs. The QQMNCs could be a promising option for fracture nonunion.Cite this article: K. Mifuji, M. Ishikawa, N. Kamei, R. Tanaka, K. Arita, H. Mizuno, T. Asahara, N. Adachi, M. Ochi. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017;6: 489–498. DOI: 10.1302/2046-3758.68.BJR-2016-0338.R1.

Impacts of maternal nutrition on uterine and placental vascularity and mRNA expression of angiogenic factors during the establishment of pregnancy in beef heifers.

We hypothesized that maternal nutrient restriction starting at the time of breeding would influence placental vascular development and gene expression of angiogenic factors during the first 50 d of gestation in beef heifers. Commercial Angus crossbred heifers (n = 49) were maintained on a total mixed ration and supplemented with dried distillers grains with solubles. All heifers were subject to 5-d CO-Synch + CIDR estrous synchronization protocol, AI to a single Angus sire, and randomly assigned to dietary treatments. One half were assigned to control diet (CON) targeted to gain 0.45 kg/d and the remaining half were assigned to restricted diet (RES), which received 60% of CON. Heifers were subjected to ovariohysterectomy on d 16, 34, or 50 of gestation. Utero-placental tissues were obtained from the uterine horns ipsilateral and contralateral to the corpus luteum and separated into maternal caruncle (CAR); maternal endometrium, inter-caruncle (ICAR), and fetal membranes (FM). After collection, all tissues were snap frozen and stored at –80°C. There were no treatment × stage of gestation interactions (P >0.13) on the mRNA expression of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS). Heifers on CON treatment had greater (P = 0.03) expression of VEGF compared with RES heifers in NP-ICAR. On d 50 expression of eNOS was increased (P = 0.05) compared with d 16 in P-CAR. Expression of eNOS mRNA was decreased (P = 0.04) on d 16 compared with d 34 and 50 in CON heifer. Gene expression of eNOS was increased (P < 0.001) in the pregnant uterine horn compared with the NP uterine horn on d 34 and 50. Expression of eNOS was also increased (P < 0.003) on d 34 and 50 in the pregnant uterine horn compared with FM. There was a maternal nutritional plane × stage of gestation interaction (P = 0.01) on the vascular ratio (vascular volume/tissue volume) in maternal tissues. The RES heifers had a greater vascular ratio on d 16 compared with d 34 and 50; whereas, CON heifers had a greater vascular ratio on d 34 compared with d 16 and 50. In the NP uterine horn, there was also an increase (P = 0.02) in vascular volume of FM from CON heifers compared with FM from RES heifers. We conclude that maternal nutrient restriction did alter both vascularity and mRNA expression of angiogenic factor in utero-placental tissues during the establishment of pregnancy in first parity beef heifers.

Notch-independent RBPJ controls angiogenesis in the adult heart.

Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury.

Schwann cells promote endothelial cell migration

Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.

Co-transplantation of endothelial progenitor cells and pancreatic islets to induce long-lasting normoglycemia in streptozotocin-treated diabetic rats.

Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3–5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards.

Mouse strain influences angiogenic response to dextran sodium sulfate–induced colitis

BackgroundAngiogenesis is a known pathologic factor in chronic inflammatory diseases. Regarding the murine dextran sodium sulfate (DSS) colitis model, different mouse strains produce variable clinical and inflammatory responses. We hypothesize that DSS colitis applied to diverse mouse strains will similarly elevate colonic microvessel density in parallel with inflammation, but will do so with different angiogenic profiles. Materials and methodsWe induced DSS colitis in 129S2/SvPas, BALB/c, and C57BL/6 mice, then performed histologic and molecular analysis at day 7 to evaluate colonic inflammation and angiogenesis. ResultsInflammation and microvessel density were similarly increased in DSS groups. The C57BL/6 cohort mounted a more severe colitis with 25% weight loss and greater colonic ulceration. Gene expression of angiogenic factors at baseline and in colitis groups were widely variable among strains. BALB/c mice exhibited higher angiogenic gene expression in control and DSS groups compared with other strains, specifically platelet-derived growth factor, angiopoietin-1, angiopoietin-1 (Ang-2), vascular endothelial growth factor receptor, and PDGF receptor. When evaluating the effect of DSS relative to controls, BALB/c mice were not significantly affected. 129S2/SvPas mice exhibited broad suppression of growth factors, significantly platelet-derived growth factor, Ang-2, and PDGF receptor. In contrast, C57BL/6 mice displayed increased gene expression, especially for angiopoietin-1 and Ang-2. ConclusionsGenetic heterogeneity influences the angiogenic profile elicited by DSS colitis. We demonstrate that within a model of murine colitis, mouse strain significantly affects inflammation-associated angiogenesis. These results may impact strain selection when using a colitis model focusing on inflammation and angiogenesis. Future studies to further define the angiogenesis pathway and potentially alter the disease course with targeted antiangiogenics are warranted.

An Overview of Molecular and Cellular Mechanisms Associated with Porcine Pregnancy Success or Failure

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.

Increased endometrial placenta growth factor (PLGF) gene expression in women with successful implantation

To analyze the vascularization of the endometrium via hysteroscopy and to assess its correlation with angiogenic factor gene expression and embryo implantation rate. Cross-sectional study. Public university hospital. Patients undergoing hysteroscopy for supposed infertility. Endometrial quality assessment according to Sakumoto-Masamoto, performed in the early secretory phase of the cycle. Collection of an endometrial tissue biopsy. RNA extraction, reverse transcription, and determination of gene expression of angiogenesis- and implantation-relevant factors using quantitative polymerase chain reaction. Retrieval of pregnancy information from the medical records. Good quantity/quality RNA with infertility history was obtained from 63 participating women. Those with a "good" endometrium and subsequent pregnancy showed increased gene expression for placenta growth factor when compared with patients with a "bad" endometrium and who did not succeed with pregnancy to date. Nonpregnant women with a "good" endometrium presented an intermediate result. No significant differences were observed for several other genes tested, but trends in the same direction were observed. This study demonstrates for the first time that endometrial PLGF expression corresponds to the hysteroscopic appearance of the endometrium, and therefore has potential as a clinically relevant prognosticator for infertility treatment success.