mRNA Expression Of Angiogenic Factors Research Articles

Conditional Mutation of Hand1 in the Mouse Placenta Disrupts Placental Vascular Development Resulting in Fetal Loss in Both Early and Late Pregnancy.

Congenital heart defects (CHD) affect approximately 1% of all live births, and often require complex surgeries at birth. We have previously demonstrated abnormal placental vascularization in human placentas from fetuses diagnosed with CHD. Hand1 has roles in both heart and placental development and is implicated in CHD development. We utilized two conditionally activated Hand1A126fs/+ murine mutant models to investigate the importance of cell-specific Hand1 on placental development in early (Nkx2-5Cre) and late (Cdh5Cre) pregnancy. Embryonic lethality occurred in Nkx2-5Cre/Hand1A126fs/+ embryos with marked fetal demise occurring after E10.5 due to a failure in placental labyrinth formation and therefore the inability to switch to hemotrophic nutrition or maintain sufficient oxygen transfer to the fetus. Labyrinthine vessels failed to develop appropriately and vessel density was significantly lower by day E12.5. In late pregnancy, the occurrence of Cdh5Cre+;Hand1A126fs/+ fetuses was reduced from 29% at E12.5 to 20% at E18.5 and remaining fetuses exhibited reduced fetal and placental weights, labyrinth vessel density and placenta angiogenic factor mRNA expression. Our results demonstrate for the first time the necessity of Hand1 in both establishment and remodeling of the exchange area beyond early pregnancy and in patterning vascularization of the placental labyrinth crucial for maintaining pregnancy and successful fetal growth.

Mendelian randomization and experimental IUGR reveal the adverse effect of low birth weight on lung structure and function

Intrauterine growth restriction (IUGR) and low birth weigth (LBW) are risk factors for neonatal chronic lung disease. However, maternal and fetal genetic factors and the molecular mechanisms remain unclear. We investigated the relationship between LBW and lung function with Mendelian randomisation analyses and studied angiogenesis in a low protein diet rat model of IUGR. Our data indicate a possible association between LBW and reduced FEV1 (p = 5.69E−18, MR-PRESSO) and FVC (6.02E-22, MR-PRESSO). Complimentary, we demonstrated two-phased perinatal programming after IUGR. The intrauterine phase (embryonic day 21) is earmarked by a reduction of endothelial cell markers (e.g. CD31) as well as mRNA expression of angiogenic factors (e.g., Vegfa, Flt1, Klf4). Protein analysis identified an activation of anti-angiogenic mTOR effectors. In the postnatal phase, lung capillaries (< 20 µm) were significantly reduced, expression of CD31 and VE-Cadherin were unaffected, whereas SMAD1/5/8 signaling and Klf4 protein were increased (p < 0.01). Moreover, elevated proteolytic activity of MMP2 and MMP9 was linked to a 50% reduction of lung elastic fibres. In conclusion, we show a possible link of LBW in humans and reduced lung function in adulthood. Experimental IUGR identifies an intrauterine phase with inhibition of angiogenic signaling, and a postnatal phase with proteolytic activity and reduced elastic fibres.

Buyang-Huanwu decoction combined with enriched environment on the abnormal activation of astrocytes and the expression of angiogenic factors in rats with cerebral ischemia

Objective To observe the effects of Buyang-Huanwu decoction (BYHWD) combined with enriched environment (EE) on the abnormal activation of astrocytes and expression of angiogenic factors in rats with cerebral ischemia. Methods The rat model of permanent middle cerebral artery occlusion (pMCAO) was established by using suture method. Male SD rats were randomly divided into sham, model, EE, BYHWD and BYHWD+EE group. Rats in the BYHWD and BYHWD+EE groups were intragastrically adminstratered with BYHWD (16.1g/kg) 24h after MCAO and once daily for consecutive 15 days. Accordingly, rats in the sham, model, and EE groups were adminstratered with the sam volume normal saline. Rats treated with EE and BYHWD+EE were housed in the enriched environment 12 h every day. The EE, BYHWD and BYHWD+EE group rats were given relative treatment continuously for 15 days. Beam walking test was performed to examine the motor function of rats. Next, the HE staining was conducted to detect the pathological alterations of the brain. Immunohistochemical staining with DARPP-32 and GFAP were applied to respectively observe the changes of multiple spinous neurons and activation of astrocytes in the striatum. Subsequently, the mRNA expressions of angiogenic factors including VEGF/VEGFR2 and Ang1/Ang2 were detected by RT-PCR. Results The rats treated with BYHWD+EE revealed markedly elevated beam walking scores on the 7th and 15th day after MCAO compared with model group (P<0.01). Compared with the model, BYHWD+EE treatment significantly increased the number of neurons (31.08 ± 8.06 vs. 19.00 ± 9.12, P<0.01), elevated the number of DARPP-32-positive cells (975.3 ± 589.68 vs. 271.25 ± 164.98) and integral optical density (18 621.87 ± 6 996.64 vs. 4 071.60 ± 2477.27) of DARPP-32 (P<0.01), suppressed the expression of GFAP (6 094.07 ± 3 211.74 vs. 10 002.91 ± 8 430.34, P<0.01) and up-regulated the mRNA levels of VEGF (1.59 ± 0.80 vs. 0.77 ± 0.33), VEGFR2 (1.58 ± 0.88 vs. 0.70 ± 0.37), Ang1 (1.58 ± 0.52 vs. 0.84 ± 0.13), and Ang2 (1.25 ± 0.25 vs. 0.90 ± 0.22) in the striatum (P<0.05 or P<0.01). Conclusions The Buyang-Huanwu decoction combined with enriched environment exerted synergistic effect on ischemic rats and promoted motor function by inhibiting the excessive activation of astrocytes and improving the expression of angiogenic factors. Key words: Bu Yang Huan Wu Tang; Brain Ischemia; Enriched environment; Astrocyte; Angiogenic; Rats

Maternal nutrient restriction alters uterine artery hemodynamics and placentome vascular density in Bos indicus and Bos taurus.

The objective was to examine uterine artery blood flow (UBF) as well as macroscopic and microscopic placentome vascular density in nutrient-restricted Angus and Brahman heifers. Angus (n = 6) and Brahman (n = 6) heifers were bred to a single sire and pregnancy confirmed at 30-d postbreeding. Heifers were randomly assigned to 1 of 2 dietary treatments consisting of 100% (control-fed; CON; n = 6) or 60% (total nutrient-restricted; RES; n = 6) based from net energy requirements for gestating heifers. Nutritional treatments were imposed from days 50 to 180 of gestation. On day 175 of gestation, UBF was collected ipsilateral and contralateral to the conceptus via Doppler ultrasonography. Heifers underwent Cesarean sections for collection of 2 adjacent placentomes on day 180 of gestation. The primary cotyledonary artery of 1 placentome was perfused with Alexa Fluor 647 Con A conjugate to examine macroscopic cotyledonary vascular density via an in vivo imaging system. The second placentome was fixed for microscopic immunofluorescence labeling of capillaries and separated into maternal (caruncle) and fetal (cotyledon) components for determination of angiogenic factor mRNA expression. Main effects of nutritional treatment and breed are reported in the absence of a significant nutritional treatment by breed interaction. Ipsilateral UBF was decreased (P < 0.05) by 48% in RES vs. CON, whereas breed did not influence ipsilateral UBF. Contralateral UBF was not different between nutritional treatments; however, contralateral UBF was decreased (P < 0.05) by 63% in Brahman vs. Angus cattle. Macroscopic cotyledonary vascular density was increased (P < 0.05) by 36% in RES vs. CON and 82% in Brahman vs. Angus heifers. Percent capillary area and capillary perimeter were increased (P < 0.05) in RES vs. CON and increased (P < 0.05) in Brahman vs. Angus heifers. Dietary treatments did not alter angiogenic factor expression; however, transcript abundance of caruncle and cotyledon ANGP1, FLT1, and KDR was increased (P < 0.05) in Brahman vs. Angus heifers. In summary, these data indicate compensatory responses in macroscopic and microscopic placentome blood vessel density during maternal nutrient restriction-induced reductions in UBF. Moreover, a greater macroscopic density of cotyledonary blood vessels was observed in Brahman vs. Angus heifers.

Recombinant human soluble thrombomodulin as an anticoagulation therapy improves recurrent miscarriage and fetal growth restriction due to placental insufficiency – The leading cause of preeclampsia

IntroductionPlacental insufficiency is one of the major risk factors for growth restriction and preeclampsia. The aim of this study is to investigate whether recombinant human Thrombomodulin(r-TM) improves fetal conditions and physiological outcomes. MethodsWe used CBA/J × BALB/C mice as a control and CBA/J × DBA/2 mice – a well-studied model of recurrent spontaneous miscarriage. Pregnant mice received daily subcutaneous injections of r-TM or saline from day 0–15. The fetal resorption rate, fetal weight, and litter size were calculated at day 15. Additionally, we analyzed the mRNA expression of angiogenic factors and the concentration of soluble Flt-1 (sFlt-1) using the ELISA kit. ResultsThe rate of fetal resorption in CBA/J × DBA/2 mice treated with r-TM was significantly lower compared with mice without r-TM treatment. Additionally, fetal weight and litter size were also significantly higher in the r-TM treated mice. Fibrinogen deposition in the labyrinth area of the CBA/J × DBA/2 mice treated with r-TM was significantly lower compared with deposits in the mice untreated with r-TM. As well, r-TM significantly increased the gene expression level of VEGF and Flt-1 mRNA in the placentas of the CBA/J × DBA/2 mice. r-TM treatment also significantly decreased the production of sFlt-1 protein in the placentas of preeclampsia-like diseased mice. Conclusionr-TM as an anticoagulation therapy has the potential for the medical treatment of recurrent miscarriage and fetal growth restriction due to improved angiogenic factors. Additionally, r-TM treatment has the potential for the recovery of preeclampsia.

M1 macrophages are involved in folliculogenesis and M2 macrophages play an essential role for successful implantation

Early administration of low dose acetyl salicylic acid (Aspirin) in high risk women reduces the risk of early onset preeclampsia. This study aims to investigate the effect of aspirin on trophoblast integration and the its effect on angiogenic and invasive pathways in an in-vitro model of preeclampsia. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells were co-cultured with the endothelial networks with/without TNF-a (0.5 ng/mL) and/or aspirin (0.1 mM) for 24 h. Fluorescent images were captured and quantified by Image J to examine the effects of TNF-a and aspirin on the trophoblast-endothelial integration. Conditioned media were collected to measure free VEGF, PlGF and sFlt-1 by ELISA and PGF1a by Enzyme immunoassay (EIA). Cells were retrieved to examine mRNA expression of angiogenic factors (VEGF, PlGF and sFlt-1), invasion markers (MMP-2 and TIMP-1), endothelial cell activation markers (E-selectin and VCAM), eNOS and cyclooxygenase (COX)-2 by quantitative PCR. Aspirin reversed the inhibitory effect of TNF-a on trophoblast cell integration into endothelial cellular networks. TNF-a increased PGF1a production (128 ± 11%, p < 0.05), whilst aspirin reversed the TNF-a effect on PGF1a production (19 ± 4%, p < 0.01). TNF-a decreased the mRNA expression of PlGF, eNOS, MMP-2 and TIMP-1, and stimulated COX2, E-selectin and VCAM mRNA expression. Aspirin did not reverse the TNF-a effect on these molecules. Aspirin improves trophoblast cell integration into endothelial cellular networks by inhibiting the effect of TNF-a via PGI2 with no significant effect on antiangiogenic, invasive or endothelial activation markers.

The effect of acetyl salicylic acid (Aspirin) on trophoblast-endothelial interaction in vitro

Early administration of low dose acetyl salicylic acid (Aspirin) in high risk women reduces the risk of early onset preeclampsia. This study aims to investigate the effect of aspirin on trophoblast integration and the its effect on angiogenic and invasive pathways in an in-vitro model of preeclampsia. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells were co-cultured with the endothelial networks with/without TNF-a (0.5ng/mL) and/or aspirin (0.1mM) for 24h. Fluorescent images were captured and quantified by Image J to examine the effects of TNF-a and aspirin on the trophoblast-endothelial integration. Conditioned media were collected to measure free VEGF, PlGF and sFlt-1 by ELISA and PGF1a by Enzyme immunoassay (EIA). Cells were retrieved to examine mRNA expression of angiogenic factors (VEGF, PlGF and sFlt-1), invasion markers (MMP-2 and TIMP-1), endothelial cell activation markers (E-selectin and VCAM), eNOS and cyclooxygenase (COX)-2 by quantitative PCR. Aspirin reversed the inhibitory effect of TNF-a on trophoblast cell integration into endothelial cellular networks. TNF-a increased PGF1a production (128±11%, p<0.05), whilst aspirin reversed the TNF-a effect on PGF1a production (19±4%, p<0.01). TNF-a decreased the mRNA expression of PlGF, eNOS, MMP-2 and TIMP-1, and stimulated COX2, E-selectin and VCAM mRNA expression. Aspirin did not reverse the TNF-a effect on these molecules. Aspirin improves trophoblast cell integration into endothelial cellular networks by inhibiting the effect of TNF-a via PGI2 with no significant effect on antiangiogenic, invasive or endothelial activation markers.

A synergistic negative effect of gestational smoke-exposure and small litter size on rat placental efficiency, vascularisation and angiogenic factors mRNA expression.

Smoking increases the risk of pregnancy complications such as spontaneous abortion and low birth weight (LBW). By cigarette smoke exposure (gestational day, GD3-17), normal-litter-size pregnancy with low birth weight (NP-LBW) and small-litter-size pregnancy with normal birth weight (SP-NBW) in rats were induced. The placental weight in SP-NBW was twice the weight of the normal in contrast with the smaller placenta in NP-LBW. Compared with the normal, placental efficiency (expressed as fetus-to-placenta weight ratio) and placental vascularisation were significantly decreased in smoke exposed placentas with more obvious decrease in SP-NBW. For NP-LBW, decreased placental vascularisation was due to decreased labyrinth vascularisation which was caused by both decreased number density and diameter of fetal capillary. For SP-NBW, decreased placental vascularisation was due to reduced proportion of labyrinth in placenta and decreased labyrinth vascularisation which was caused by decreased fetal capillary number density. Real time RT-PCR analysis showed a tendency for decreased placental mRNA level of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang1) and tyrosine kinase receptor-2 (Tie2) in NP-LBW(P<0.1), and the tendency became obvious in SP-NBW(P<0.05). A tendency for decreased placental mRNA level of fms-like tyrosine kinase-1(Flt1) and angiopoietin-2 (Ang2) was also observed in SP-LBW(P<0.1). Our data demonstrated the synergistic negative effect of gestational smoke-exposure and small litter size on placental efficiency, placental vascularisation and placental angiogenic growth factor mRNA expression in rat.

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.

Thermotherapy Application Increases The mRNA Expression of Angiogenic Factors in Human Skeletal Muscle

Treatment with thermotherapy (TT) reduces the clinical symptoms and improves exercise tolerance in patients with several chronic disease conditions, but the molecular mechanisms by which this therapy exerts its benefits remain poorly defined. We tested the hypothesis that a single session of TT increases systemic and skeletal muscle levels of angiogenic mediators in young individuals. Forty‐one healthy young subjects were recruited to participate in two separate studies (Study 1, n=18 and Study 2, n=23). A water‐circulating garment that covered both legs was used to apply TT or a control intervention on both studies. Water at 48°C was perfused through the garment for 90 min for TT application, while thermoneutral water (33°C) was used in the control intervention. In study 1, participants completed two experimental sessions (TT and control), at least 72 hrs apart, in a randomized, cross‐over design. Blood samples were taken before and 30 and 120 min following the completion of the trials for the determination of the percentage of circulating pro‐angiogenic cells (multiparametric flow‐cytometry) and the levels of inflammatory and angiogenic factors (multiplex immunoassays). In study 2, participants were randomly allocated to either a TT‐treated group (n=11) or to a control group (n=12). The treatments (TT or control) were applied for 90 min as described above. Biopsies were taken from the vastus lateralis muscle prior to and 30 and 120 min following the interventions. The mRNA expression of inflammatory and angiogenic factors and members of the heat shock protein family was determined using real‐time polymerase chain reaction. Application of TT for 90 min in study 1 had no effect on circulating levels of angiogenic cytokines and the percentage of bone marrow‐derived pro‐angiogenic cells (CD34+CD133+), but the levels of vasoconstrictor endothelin‐1 were lower 30 min after TT when compared to the control intervention (p&lt;0.05). Conversely, in skeletal muscle (Study 2), TT evoked a transient increase in the mRNA expression of several important angiogenic factors and heat shock proteins. When compared to the control group, the change in mRNA expression from baseline of vascular endothelial growth factor, angiopoietin‐2, chemokines CCL2 and CX3CL1, the ratio of angiopoietin‐2/angiopoietin‐1 and the angiostatic factor platelet factor‐4 was higher (p&lt;0.05) 30 min after the intervention in the TT‐treated group. Likewise, the expression of members of the heat shock protein family was up‐regulated 30 min after TT compared to control (p&lt;0.05). The expression of the angiostatic transcription factor FoxO1 was lower in the TT group at 120 min following the end of intervention period (p=0.03). These novel findings indicate that TT is a simple, non‐invasive strategy to activate local angiogenic signaling and possibly promote vascular growth in skeletal muscle.

The anti-angiogenic herbal extract from Melissa officinalis inhibits adipogenesis in 3T3-L1 adipocytes and suppresses adipocyte hypertrophy in high fat diet-induced obese C57BL/6J mice

Ethnopharmacological relevanceMelissa officinalis L. (Labiatae; lemon balm) has been used traditionally and contemporarily as an anti-stress herb. Current hypotheses suggest that not only chronic stress promotes angiogenesis, but angiogenesis also modulates adipogenesis and obesity. Because the herbal extract ALS-L1023 from M. officinalis L. (Labiatae; lemon balm) has an anti-angiogenic activity, we hypothesized that ALS-L1023 could inhibit adipogenesis and adipocyte hypertrophy. Materials and methodsALS-L1023 was prepared by a two-step organic solvent fractionation from M. officinalis. The effects of ALS-L1023 on adipogenesis in 3T3-L1 adipocytes and adipocyte hypertrophy in high fat diet (HFD)-fed obese mice were measured using in vivo and in vitro approaches. ResultsALS-L1023 inhibited angiogenesis in a dose-dependent manner in the HUVEC tube formation assay in vitro. Treatment of cells with ALS-L1023 inhibited lipid accumulation and adipocyte-specific gene expression caused by troglitazone or MDI differentiation mix. ALS-L1023 reduced mRNA expression of angiogenic factors (VEGF-A and FGF-2) and MMPs (MMP-2 and MMP-9) in differentiated cells. In contrast, mRNA levels of angiogenic inhibitors (TSP-1, TIMP-1, and TIMP-2) increased. Protease activity, as measured by zymography, showed that activity of MMP-2 and MMP-9 decreased in ALS-L1023-treated cells. ALS-L1023 also inhibited MMP-2 and MMP-9 reporter gene expression in the presence of the MMP inducer phorbol 12-myristate 13-acetate. An in vivo study showed that ALS-L1023 not only decreased adipose tissue mass and adipocyte size, but also reduced mRNA levels of adipose tissue angiogenic factors and MMPs in HFD-fed obese mice. ConclusionsThese results suggest that the anti-angiogenic herbal extract ALS-L1023 suppresses adipogenesis and adipocyte hypertrophy, and this effect may be mediated by inhibiting angiogenesis and MMP activities. Thus, by curbing adipogenesis, anti-angiogenic ALS-L1023 yields a possible therapeutic choice for the prevention and treatment of human obesity and its associated conditions.

Local delivery of COMP-angiopoietin 1 accelerates new bone formation in rat calvarial defects.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent in bone reconstruction. However, the potential of COMP-Ang1 to regenerate impaired bone and induce new bone formation has not been completely explored. In this study, male Sprague-Dawley rats underwent calvarial defect surgery and divided into two groups: scaffold treatment alone (control group) and COMP-Ang1-impregnated scaffold (COMP-Ang1 group). According to live micro-CT and histological analyses, the COMP-Ang1 group showed greater new bone formation and maturation than did the control both four and eight weeks after surgery. The values of bone volume, bone mineral density, and bone surface were also higher in the COMP-Ang1 group than in the control at the same weeks after surgery. In addition, the delivery of COMP-Ang1 facilitated significantly the expression of osteoblast-specific markers such as runt-related transcription factor 2 (p < 0.001), osterix (p < 0.001), bone morphogenetic protein-2 (p < 0.001), alkaline phosphatase (p < 0.01), osteocalcin (p < 0.001), and type I collagen (p < 0.05) in newly formed bone, compared with the control. Immunohistochemistric assay supported the COMP-Ang1-facilitated induction of bone-specific markers. Furthermore, COMP-Ang1 augmented the mRNA expression of angiogenic factors, especially of platelet endothelial cell adhesion molecule 1, stromal cell-derived factor 1, and Tie-2 in the defect site. Our current findings demonstrate for the first time that a local delivery of recombinant COMP-Ang1 promotes bone formation in calvarial defects, which is coupled with enhanced angiogenesis and chemoattraction.

10.6 Influence of prenatal adenovirus-VEGF gene therapy on mRNA expression of angiogenic related genes in fetal and postnatal ovine intestine

Maternal uterine artery adenovirus VEGF (Ad. VEGF-A165) gene therapy has increased pre- and early postnatal growth velocity in an overnourished adolescent ovine model of fetal growth restriction. Our objective was...

PFM.01 Influence of prenatal adenovirus-VEGF gene therapy on mRNA expression of angiogenic related genes in fetal and postnatal ovine intestine

Maternal uterine artery adenovirus VEGF (Ad. VEGF-A165) gene therapy has increased pre- and early postnatal growth velocity in an overnourished adolescent ovine model of fetal growth restriction. Our objective was...

Adipose-derived stem cells promote angiogenesis and tissue formation for in vivo tissue engineering.

Adult mesenchymal stem cells secrete a variety of angiogenic cytokines and growth factors, so we proposed that these paracrine mechanisms may be used to promote vascularization and growth for tissue engineering in vivo. We tested whether or not human adipose-derived stem cells (ASCs) promote tissue formation in rats. ASCs were evaluated in vitro for mRNA expression of angiogenic factors, including the vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 (IL-8), and stromal cell-derived factor-1 (SDF-1) and proliferative activity on human microvascular endothelial cells. For in vivo analysis, CM-DiI-labeled ASCs were implanted with a rat cardiac extracellular matrix (ECM) extract-derived hydrogel into a chamber with a femoral arteriovenous loop in the groin of male nude rats for 7 days. Vascularization in newly generated tissue was estimated by histomorphometry after endothelial nitric oxide synthase (eNOS) immunostaining. ASCs expressed growth factor mRNA and produced an angiogenic activity in vitro. After implantation, ASCs survived, but remained suspended in the ECM and relatively few were incorporated into the newly formed tissue. The volume of newly generated tissue was significantly higher in chambers containing ASCs and it was enriched with vasculature when compared with the ECM alone. We conclude that human ASCs promote tissue growth and angiogenesis in the rat vascularized chamber, thereby showing promise for tissue-engineering applications for regenerative therapy.

Impacts of linseed meal and estradiol-17β on cellularity, angiogenic and vasoactive factor mRNA expression, and vascularity of the uterus in ovariectomized ewes

Ilse, B. R., O'Neil, M. R., Lardy, G. P., Reynolds, L. P. and Vonnahme, K. A. 2012. Impacts of linseed meal and estradiol-17β on cellularity, angiogenic and vasoactive factor mRNA expression, and vascularity of the uterus in ovariectomized ewes. Can. J. Anim. Sci. 92: 297–306. The objective of the current study was to determine the estrogenic potential of the phytoestrogen secoisolariciresinol diglycoside (SDG) found in linseed meal (LSM) on uterine cell proliferation, vascularity, and angiogenic factor mRNA expression. Ovariectomized ewes (n=48) were fed a diet containing 12.5% LSM for 0, 1, 7, or 14 d and implanted with estradiol-17β (E2) for 0, 6, or 24 h before tissue collection. There was an interaction of LSM×E2 on uterine mass (grams; P=0.03; percentage change; P&lt;0.003). Uterine mass increased (P≤0.02) after 24 h of E2 exposure on days 1, 7, and 14 of LSM feeding, with the greatest mass occurring in ewes exposed to E2 for 24 h and 1 d LSM feeding. Regardless of days fed LSM, after 24 h of E2 exposure uterine mass was greatest. The greatest percentage increase in uterine mass occurred in ewes exposed to E2 for 24 h and fed 1 d of LSM. Cell proliferation within the uterine luminal epithelium was greatest (P&lt;0.01) with 24 h of E2 exposure compared with 0 h and 6 h. When expressed as the percentage change in uterine cell proliferation, feeding LSM for 14 d negated these effects. Only length of E2 exposure impacted vascularity with capillary number density at 6 h of E2 exposure being greater (P=0.02) than at 24 h. While mRNA expression of several angiogenic factors was influenced by E2, there was a LSM×E2 interaction (P≤0.03) only on vascular endothelial growth factor receptor 2 and fibroblast growth factor receptor 2C. It appears that growth and angiogenesis of E2 sensitive tissues may be influenced by the duration of LSM feeding.

Critical Role of TNF-α-Induced Macrophage VEGF and iNOS Production in the Experimental Corneal Neovascularization

We evaluated the roles of tumor necrosis factor (TNF)-α in alkali-induced corneal neovascularization (CNV). CNV was induced by alkali injury and compared in wild-type (WT) BALB/c mice, and TNF receptor 1-deficient (TNF-Rp55 KO) counterparts, or in mice treated with TNF-α antagonist and recombinant TNF-α. Angiogenic factor expression and leukocyte accumulation in the early phase after injury were quantified by real-time PCR and immunohistochemical analysis, respectively. Alkali injury augmented the intraocular mRNA expression of TNF-α and its receptor, together with a transient macrophage and neutrophil infiltration. Compared to WT mice, TNF-Rp55 KO mice exhibited reduced CNV. Intraocular F4/80-positive macrophages and Ly-6G-positive neutrophils infiltration did not change in KO mice compared to WT mice after the injury. Alkali injury induced a massively increased intraocular mRNA expression of angiogenic factors, including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), interleukin (IL)-6, E-selectin, and intercellular adhesion molecule (ICAM)-1 in WT mice, whereas these increments were retarded severely in KO mice. Immunofluorescence analysis demonstrated that F4/80-positive cells expressed VEGF and iNOS. Moreover, TNF-α enhanced VEGF and iNOS expression by peritoneal macrophage from WT, but not KO mice. Topical application of TNF-α antagonist reduced CNV, while topical application of recombinant TNF-α enhanced it. TNF-Rp55-KO mice exhibited impaired alkali-induced CNV through reduced intracorneal infiltrating macrophage VEGF and iNOS expression.

Supranutritional selenium increases mammary gland vascularity in postpartum ewe lambs

Objectives were to determine the effects of maternal dietary supranutritional Se and nutritional plane during gestation on capillary surface density, capillary area density, and angiogenic factor expression in the developing mammary gland of primiparous ewes. Selenium treatments were initiated at breeding [adequate Se (ASe; 9.5μg/kg of body weight) vs. high Se (HSe; 81.8μg/kg of body weight)] and nutritional planes at d 50 of gestation [Low, 60%; moderate (Mod), 100%; and High, 140% of requirements). Mammary glands were collected within 24h postpartum. Vascular development was assessed in the glandular portion of the mammary gland. Vascularity was determined for mammary tissue with the following measurements taken: the cross-sectional capillary area density (total capillary area as a proportion of tissue area) and capillary surface density (CSD; total capillary circumference per unit of tissue area). High-Se ewes had greater capillary surface and area densities compared with ASe ewes. A tendency existed for an Se × plane of nutrition interaction for CSD with maternal diet not affecting CSD in HSe ewes, but Low ewes had a decreased CSD compared with Mod ewes, with High being intermediate in ASe ewes. Moreover, HSe-Low and HSe-High ewes had increased CSD compared with ASe-Low and ASe-High, respectively. Although Se status did not influence angiogenic factor mRNA expression, mammary glands from Low ewes tended to have increased VEGF and FLT1 mRNA expression compared with High ewes, with Mod being intermediate. Maternal plane of nutrition did not affect mammary gland glutathione peroxidase activity, but it was increased in HSe compared with ASe ewes. Increased mammary capillary nutrient exchange area may contribute to previously observed changes in colostrum quality.

Abstract 222: Differential p73 isoform expression profile in non-Hodgkin's lymphoma with 1p36 chromosomal abnormality modulates angiogenic phenotype

Abstract Alteration of tumor suppressor gene function often leads to imbalance in expression of angiogenic and angiostatic factors facilitating disease progression. TP73, a member of the p53 tumor suppressor gene family, regulates many important cellular pathways and has been shown to be abnormally expressed in non-Hodgkin's lymphoma (NHL). TP73 gene function is controlled by harmony between two isoforms with opposing functions, the full length TAp73 protein (pro-apoptotic) and the NH2-terminally truncated ΔNp73 protein (anti-apoptotic through antagonizing both TAp73 and p53). We have observed differential ΔNp73 up-regulation and increase in ΔNp73:TAp73 expression ratio among NHL cases with 1p36 chromosomal disruption as compared to NHL cases without 1p36 disruption, which is associated with aggressiveness. To decipher whether deregulated p73 isoforms expression modulates the angiogenic potential in NHL we examined the mRNA expression of angiogenic factors (VEGF-A, FGF-2, and angiogenic chemokine CXCL-8) and angiostatic factors (TSP-1 and Endostatin) in NHL cases with and without chromosome 1p36 abnormality by real-time PCR. We observed higher levels of expression of angiogenic factors (VEGF-A, FGF-2, and CXCL-8) and lower levels of angiostatic factors (TSP-1 and Endostatin) in NHL cases with 1p36 abnormality as compared to NHL cases without 1p36 abnormality. Next, we performed a non-parametric bivariate correlation analysis (Kendall's tau_b and Spearman's rho) to examine whether ΔNp73:TAp73 expression ratio correlates with the expression of angiogenic and angiostatic factors. We observed a significant correlation between ΔNp73:TAp73 expression ratio with the expression of VEGF-A (r=0.900, p&amp;lt;0.001), FGF-2 (r=0.620, p&amp;lt;0.05) and TSP-1 (r=−0.873, p&amp;lt;0.001). To further elucidate the relationship between differential p73 isoforms expression and the expression pattern of angiogenic and angiostatic factors, we stably transfected an NHL cell line with 1p36 disruption, Granta-519 (TAp73 non expressor) with a wild type TAp73 mammalian expression vector. We observed an up-regulation of TSP-1 and Endostatin expression among Granta-519 cells transfected with TAp73 vectors compared to isogenic control vector transfected cells. Our data highlights the molecular and functional consequences of the differential p73 isoforms expression pattern caused by 1p36 chromosomal disruption on angiogenic phenotype in NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 222. doi:10.1158/1538-7445.AM2011-222

Hypocellularity and insufficient expression of angiogenic factors in implanted autologous bone marrow in patients with chronic critical limb ischemia

The aim of this study was to identify the clinical parameters of absolutely poor-prognosis patients with chronic critical limb ischemia (AP-CLI). Sixteen no-option CLI patients with arteriosclerosis obliterans: ASO (nine) and non-ASO patients (seven) treated with bone marrow-mononuclear cell implantation (BMI) were analyzed. There were three AP-CLI patients (all ASO). The mRNA expression of several angiogenic factors in the implanted cells was analyzed in comparison with normal donor bone marrow. To observe the response of bone marrow components to hypoxia, normal bone marrow cells were cultured for 24 h in 2.5% O(2), and mRNA expression of angiogenic factors were measured. AP-CLI patients exhibited extraordinary low bone marrow cellularity as well as the percentage of CD34-positive cells. Among angiogenic factors, only VEGF expression was maintained in response to HIF-1, while other factors such as HGF, Ang-1, PLGF, and SDF-1 decreased in the implanted bone marrow cells of the patients with CLI compared to normal bone marrow cells. HIF-1 and all of the five angiogenic factors increased in vitro in response to hypoxia. Thus it is highly likely that angiogenic factors except VEGF do not respond to chronic ischemia in bone marrow in vivo. An organ-protection system against tissue ischemia may be applied for acute hypoxia, but it may be insufficient for chronic ischemia.