Expression Of Angiogenic Proteins Research Articles

Constructing Mg-phytic acid enhanced hydrogel patch with janus structure for antibacterial and angiogenic urethral repair

Due to bacteria in urine and inadequate blood supply, urethral defect repair faces great clinical difficulties. Inspired by the natural structure of the urethra, in this study, we designed a multifunctional hydrogel urethral patch with a Janus structure. The inner layer was a double-network hydrogel of polyvinyl alcohol/carboxymethyl chitosan (PVA/CMCS) enhanced by natural phytic acid (PA) and Mg2+ (Mg2+-PA/PVA/CMCS). The outer layer was composed of poly (L-lactic acid) (PLLA) nanofibers to form a hydrophobic barrier, coupling via a micelle layer to ensure interfacial bonding. The patch exhibited mechanical properties matching soft tissues, with maximum tensile strength and toughness of 1.69±0.13 MPa and 24.7±0.9 MJ/m3, respectively, under the strengthening effect of Mg2+-PA chelation. Therein, the 4 % Mg2+-PA/PVA/CMCS composite patch could maintain unilateral hydrophobicity for at least 14 days and demonstrated an antibacterial rate of 99.0 %±0.1 % against E. coli. In PBS, the patch could deliver Mg2+ for at least 7 days and stabilize the surrounding pH environment for up to 28 days. In vitro cell experiments indicated that the 4 % Mg2+-PA/PVA/CMCS composite patch could promote the human umbilical vein endothelial cell (HUVEC) proliferation and migration, with the angiogenic protein expression enhanced more than 1.2 times compared with the Control group. In conclusion, the patch constructed in this study may provide a new approach to clinical urethral injury repair.

The effectiveness of edible bird's nest in lowering VEGF, CD31, and PDGFR-β levels in diabetic retinopathy in rats with type 1 diabetes.

Diabetic eye disease, known as diabetic retinopathy (DR), is one of the problems that can arise from having high blood sugar for an extended period. This study aimed to investigate the effect of the edible bird's nest (EBN) on retinal angiogenesis in diabetic rats. The 50 rats were separated into five different groups, each containing 10 rats: control, diabetes (DM), bird's nest-fed diabetes (75 mg/kg Body weight; BW), (EBN 75), (150 mg/kg BW) (EBN 150), and glyburide (GR) for an eight-week study. H&E and Masson's trichrome staining were utilized to investigate the retinal tissue and vascular changes. The immunofluorescence study was used to detect angiogenic protein expression. The vascular corrosion cast/SEM method was also used to evaluate capillary plexus formation within the retinal layer. From histological studies, DM rats have thinning of the retinal layer. Remarkably, the retinal vessels displayed dilations resembling ruptured blood vessels. The expression of vascular endothelial growth factor (VEGF) (30.51±2.62), cluster of differentiation 31 (CD31) (28.18±0.22), and platelet-derived growth factor receptor beta (PDGFR-β) (141.67±0.97) were increased. EBN 75 exhibited some small improvements in their blood vessels and eye tissue. At a dose of 150 mg/kg BW, EBN proved to be more effective. There was a significant decrease in VEGF and CD31 expression compared with the diabetic group (p<0.001 and p<0.01, respectively). These studies have demonstrated that EBN can lower the growth levels of VEGF, CD31, and PDGFR-β, which results in a decrease in angiogenesis and a recovery from a variety of diabetic retinopathy-related diseases.

Dual‑directional effect of vinorelbine combined with cisplatin or fluorouracil on tumor growth and metastasis in metronomic chemotherapy in breast cancer

Dual‑directional effect of vinorelbine combined with cisplatin or fluorouracil on tumor growth and metastasis in metronomic chemotherapy in breast cancer

A hyaluronic acid / chitosan composite functionalized hydrogel based on enzyme-catalyzed and Schiff base reaction for promoting wound healing

The healing of full-thickness skin defect has been a clinical challenge. Hydrogels with multiple functions inspired by extracellular matrix are expected to be used as wound dressing. In this paper, dopamine-grafted oxidized hyaluronic acid was blended with quaternary ammonium chitosan to form a composite functionalized hydrogel by enzyme-catalyzed cross-linking and Schiff base reaction. The hydrogel has convenient preparation, good biocompatibility, antibacterial and antioxidant, high adhesion and self-healing properties. The results in vivo show that the hydrogel can effectively close the wound and accelerate the speed of wound healing by up-regulating the expression of angiogenic protein and promoting the distribution of collagen deposition more uniform and regular. It is expected that this composite functionalized hydrogel dressing has great potential in wound regeneration.

Abstract 11795: CXCL5 Inhibition Recovers Neovascularization in Diabetes Mellitus

Introduction: Diabetes mellitus (DM) is a chronic inflammatory disease accompanied by dysfunction of vascular endothelial cells. Vascular complications are the main causes of death in patients with DM. CXCL5 is an inflammatory chemokine that upregulated in patients with type 2 DM. However, its role in diabetic vasculopathy was not clarified. This study aimed to explore the impacts and mechanistic insights of CXCL5 in neovasculogenesis in DM. Methods: Endothelial progenitor cells (EPCs) and human aortic endothelial cells (HAECs) were used in vitro. Streptozotocin-induced diabetic mice and dbdb mice were used as type 1 and type 2 DM models. CXCL5 knockout mice were also used to generate the diabetic animal model. The hindlimb ischemia surgery, matrigel plug assay, and aortic ring assay were conducted. Flow cytometry were used to evaluate the circulating number of EPCs. Results: CXCL5 concentrations were increased in plasma and EPCs culture medium from type 2 DM patients. CXCL5 inhibition by neutralizing antibody upregulated VEGF/SDF-1 expression and improved migration and tube formation abilities in EPCs from type 2 DM patients and high glucose-treated EPCs from non-diabetic subjects as well as HAECs. Intraperitoneal injection of CXCL5 neutralizing antibody increased circulating EPC number, enhanced capillary density, and recovered the blood flow after hindlimb ischemia surgery in both type 1 and type 2 diabetic animals. Protein levels of VEGF and SDF-1 in ischemic muscle were upregulated in the CXCL5 inhibition group. Additionally, diabetic mice injected with CXCL5 neutralizing antibody showed higher hemoglobin contents and neovascular branches in the matrigel plug assay and aortic ring assay, respectively. Importantly, CXCL5 knockout diabetic mice showed improved ischemia-induced angiogenesis and higher expression of angiogenic proteins in ischemic muscle compared with wild-type diabetic mice. Conclusion: CXCL5 inhibition can improve neovasculogenesis via protecting vascular endothelial function and upregulating angiogenic protein expressions in both type 1 and type 2 DM. Further clinical studies are needed to verify that CXCL5 may be a potential therapeutic target for DM vascular complications.

Abstract 538: Local Hydrogen Sulfide Therapy Augments Angiogenesis And Ischemic Wound Healing

Introduction: Hydrogen sulfide (H 2 S) is an important signaling molecule in cellular O 2 sensing, wound healing and angiogenesis. Low H 2 S levels have been shown in diabetes and cardiovascular disease and H 2 S impairment may play a role in chronic non-healing ischemic wounds. H 2 S promotes angiogenesis in cell culture but in vivo data are lacking. We utilized a novel local H 2 S delivery method to investigate the effects of H 2 S therapy in rodents. Methods: Sprague Dawley rats (n=6-10/group) underwent dorsal myocutaneous ischemic flap wound creation under anesthesia. NaHS solution (100 μM) was delivered locally to the wound every 12 hours via implanted microtubing externalized and attached to a fluid delivery vest. Sham rats received saline solution while a third group received H 2 S blocker DL-propargylglycine (PAG). Laser speckle contrast images (LSCI) were recorded over 14 days. Flap tissue was collected for histologic analysis of tissue viability and degree of ischemic insult. Neovascularization was quantified by CD31 immunohistochemistry (IHC). VEGF protein expression was quantified by Western blot. Results: H 2 S treated flaps reperfused at a two-fold greater rate compared to sham and PAG treated flaps by LSCI (mean 12.9 flux/day vs. 6.9 flux/day, P=0.02). IHC mean microvascularity in sham flaps was 18 vessels/mm 2 compared to 47 vessels/mm 2 in H 2 S treated flaps (P=0.0092). Panniculus carnosis muscle thickness (408 μm vs. 234 μm) and myofibril count (56 vs. 38/100k μm 2 ) were preserved in H 2 S treated flaps (P=0.02, 0.04). H 2 S treatment upregulated VEGF while PAG was associated with decreased expression. Conclusion: H 2 S treated flaps have improved wound revascularization, tissue healing and angiogenic signaling. Blockade of H 2 S results in poor revascularization and decreased angiogenic protein expression. H 2 S pathways may play a critical role in the pathogenesis of impaired wound healing and could represent a potential therapeutic target for ischemic wounds.

JUND facilitates proliferation and angiogenesis of esophageal squamous cell carcinoma cell via MAPRE2 up-regulation

ObjectiveEsophageal squamous cell carcinoma (ESCC) is a globally aggressive malignant tumor. This study aimed to investigate the mechanism of JUND in ESCC development via MAPRE2. MethodsESCC cells (KYSE-450 and ECA109) were transfected with small interfering RNA (si)-JUND, si-MAPRE2, si-JUND, or pcDNA3.1-MAPRE2. JUND and MAPRE2 expression in ESCC cells was detected with quantitative real-time polymerase chain reaction and western blot. Cell counting kit-8 and 5-ethynyl-2′-deoxyuridine assays were used to determine ESCC cell proliferation. Dual-luciferase reporter gene and chromatin immunoprecipitation assays were performed to assess binding between JUND and MAPRE2. Human umbilical vein endothelial cells (HUVECs) were co-cultured with ESCC cell supernatants. Angiogenesis was assessed with an in vitro angiogenesis assay. Western blot was conducted to evaluate the expression of angiogenic proteins [vascular endothelial growth factor A (VEGFA), matrix metallopeptidase 9 (MMP-9), and angiopoietin-2 (ang2)]. ResultsThe levels of expression of JUND and MAPRE2 were high in ESCC cells. Mechanistically, JUND bound to MAPRE2 promoter and increased MAPRE2 transcription. Downregulation of JUND or MAPRE2 inhibited KYSE-450 and ECA109 cell proliferation and reduced the levels of expression of VEGFA, MMP-9, and ang2 and tube formation in HUVECs co-cultured with ESCC cell supernatants. MAPRE2 upregulation counteracted the inhibitory effects of JUND silencing on cell proliferative and angiogenic capabilities in ESCC. ConclusionsJUND promoted MAPRE2 transcription, thereby facilitating cell proliferative and angiogenic abilities in ESCC.

Exosomes from bone mesenchymal stem cells alleviate mifepristone-induced human endometrial stromal cell injury by inhibiting TLR3 via delivering miR-941.

We aim to investigate the protective effect and underlying mechanisms of BMSCs-exo on human endometrial stromal cells (HESCs) induced by mifepristone in this study. BMSCs-exo were extracted and then identified by transmission electron microscopy and western-blot assay. RT-PCR assay was used to determine the level of miR-941. MiR-941 mimics or inhibitor were transfected into BMSCs and the exosomes were extracted. Then, Cell activity, apoptosis rate, cell migration and invasion, as well as the expression of angiogenic proteins were determined in HESCs stimulated by mifepristone and BMSCs-exo. Next, Dual-luciferase reporting assay was used to verify the targeted binding of miR-941 to TLR3, and the TLR3 expression in HESCs was detected by RT-PCR and western-blot. Finally, TLR3 was overexpressed to evaluate the effects of miR-941 from BMSCs-exo on cell apoptosis, cell invasion and angiogenesis in HESCs induced by mifepristone. miR-941 was highly expressed in BMSCs-exo. Exosome miR-941 in BMSCs-exo inhibited the cell apoptosis, and promoted cell activity, cell migration, invasion as well as angiogenesis were also improved in HESCs induced by mifepristone. TLR3 was a target of miR-941, which was up-regulated in mifepristonetreated HESCs. We further found that miR-941 derived from BMSCs-exo down-regulated the expression of TLR3 in HESCs treated by mifepristone. In addition, TLR3 overexpression blocked the inhibition of miR-941 on mifepristone-induced cell apoptosis, as well as cell migration and angiogenesis in HESCs. Thus, we concluded that BMSCs-exo has protective effect on mifepristone-induced cell damage by delivering miR-941 which targeted TLR3 and regulated cell activity, migration, and angiogenesis in HESCs.

Abnormal cerebral microvascular perfusion and reactivity in female offspring of reduced uterine perfusion pressure (RUPP) mice model

Children born from women with preeclampsia have alterations in cerebral neurovascular development and a high risk for developing cognitive alterations. Because cerebral blood vessels are critical components in cerebrovascular development, we evaluated the brain microvascular perfusion and microvascular reactivity (exposed to external stimuli of warm and cold) in pups born to preeclampsia-like syndrome based on the reduction of uterine perfusion (RUPP). Also, we evaluate the angiogenic proteomic profile in those brains. Pregnant mice showed a reduction in uterine flow after RUPP surgery (−40 to 50%) associated with unfavorable perinatal results compared to sham mice. Furthermore, offspring of the RUPP mice exhibited reduced brain microvascular perfusion at postnatal day 5 (P5) compared with offspring from sham mice. This reduction was preferentially observed in females. Also, brain microvascular reactivity to external stimuli (warm and cold) was reduced in pups of RUPP mice. Furthermore, a differential expression of the angiogenic profile associated with inflammation, extrinsic apoptotic, cancer, and cellular senescence processes as the primary signaling impaired process was found in the brains of RUPP-offspring. Then, offspring (P5) from preeclampsia-like syndrome exhibit impaired brain perfusion and microvascular reactivity, particularly in female mice, associated with differential expression of angiogenic proteins in the brain tissue.

Celastrol attenuates amphiregulin expression by inhibiting MAPK signaling pathway in triple-negative breast cancer cells

BackgroundAmphiregulin (AREG) plays an essential role in the metastasis and angiogenesis of various cancers. PurposeThe objective of this study was to investigate the pharmacological effects of celastrol on AREG expression in triple-negative breast cancer (TNBC) cells. MethodsWe analyzed the prognostic value of AREG, EGF, TGFA, and HB-EGF mRNA expression in TNBC patients. Cell proliferation was analyzed by MTT assay and colony-forming assay. Levels of protein and gene expression were analyzed by ELISA, Western blotting, confocal microscopy, and real-time PCR, respectively. The expression of angiogenic proteins was analyzed by using the Human angiogenesis array kit. Cell invasiveness was analyzed by Boyden chamber assay. ResultsOur results showed that abnormal AREG induction remarkably decreased the relapse-free survival (RFS) rate of TNBC patients, whereas epidermal growth factor (EGF), transforming growth factor-alpha (TGFA), or heparin-binding EGF-like growth factor (HBEGF) demonstrated no effect on RFS rate. In addition, the growth of TNBC cells was prevented by drugs targeting EGFR such as neratinib or lapatinib. On the contrary, cell growth and invasion were enhanced by AREG treatment. Interestingly, celastrol reduced AREG expression and the growth of TNBC cells. Furthermore, we found that levels of AREG expression were reduced by a potent inhibitor of MEK1/2, binimetinib (BIN). Our results showed that celastrol significantly decreased the levels of ERK phosphorylation in TNBC cells. Finally, we observed that celastrol also suppressed the AREG-induced EGFR signaling pathway. Taken together, these results suggest that celastrol can downregulate AREG expression by inhibiting the MEK/ERK signaling pathway. In addition, the AREG-induced EGFR signaling pathway was inhibited by celastrol. ConclusionCelastrol can be an effective drug for treating TNBC by inhibiting AREG/EGFR signaling pathways.

AT2R activation increases in vitro angiogenesis in pregnant human uterine artery endothelial cells.

Angiogenesis is vital during pregnancy for remodeling and enhancing vasodilation of maternal uterine arteries, and increasing uterine blood flow. Abnormal angiogenesis is associated with decreased uteroplacental blood flow and development of pregnancy disorders such as gestational hypertension, preeclampsia, fetal growth restriction, preterm delivery, stillbirth, and miscarriage. The mechanisms that contribute to normal angiogenesis remain obscure. Our previous studies demonstrated that expression of the angiotensin type 2 receptor (AT2R) is increased while the angiotensin type 1 receptor (AT1R) is unchanged in the endothelium of uterine arteries, and that AT2R-mediated pregnancy adaptation facilitates enhanced vasodilation and uterine arterial blood flow. However, the role of AT2R in regulating angiogenesis during pregnancy has never been studied. This study examines whether or not AT2R activation induces angiogenesis and, if so, what mechanisms are involved. To this end, we used primary human uterine artery endothelial cells (hUAECs) isolated from pregnant and nonpregnant women undergoing hysterectomy. The present study shows that Compound 21, a selective AT2R agonist, induced proliferation of pregnant-hUAECs, but not nonpregnant-hUAECs, in a concentration-dependent manner, and that this C21-induced mitogenic effect was blocked by PD123319, a selective AT2R antagonist. The mitogenic effects induced by C21 were inhibited by blocking JNK—but not ERK, PI3K, and p38—signaling pathways. In addition, C21 concentration dependently increased cell migration and capillary-like tube formation in pregnant-hUAECs. The membrane-based antibody array showed that C21 increased expression of multiple angiogenic proteins, including EGF, bFGF, leptin, PLGF, IGF-1, and angiopoietins. Our qPCR analysis demonstrates that C21-induced increase in expression of these angiogenic proteins correlates with a proportional increase in mRNA expression, indicating that AT2R activates angiogenic proteins at the transcriptional level. In summary, the present study shows that AT2R activation induces angiogenesis of hUAECs in a pregnancy-specific manner through JNK-mediated pathways with associated transcriptional upregulation of multiple proangiogenic proteins.

Morin Attenuated Cerebral Ischemia/Reperfusion Injury Through Promoting Angiogenesis Mediated by Angiopoietin-1-Tie-2 Axis and Wnt/β-Catenin Pathway.

Cerebral damage following cerebral ischemia/reperfusion injury affects the neurological deficits and motor impairment of stroke patients in the long-term period. Angiogenesis, the essential process for restoration of cerebral blood flow (CBF) in the ischemic brain, promotes the recovery of neurological function following ischemia. The aim of this study was to investigate the long-term effects of morin on angiogenesis and functional outcomes in a middle cerebral artery occlusion (MCAO) and reperfusion model. Male Wistar rats were subjected to MCAO, and they were administered 30mg/kg of morin at reperfusion via i.p. injection daily for 14days. Fourteen days after I/R injury, the rats were evaluated for the brain damage, and angiogenic factors involved in Ang1/Tie-2 and Wnt/β-catenin signaling. In addition, at 1, 7, and 14days after reperfusion, rotarod and pole tests were performed to investigate the functional recovery. We found morin significantly reduced the infarct size, blood-brain barrier (BBB) leakage, and apoptotic cells at 14days after I/R injury. It also promoted angiogenesis via boosting the expression of angiogenic proteins, such as angiopoietin 1 (Ang1), Tie-2, Wnt3α, β-catenin, and cyclin D1. Morin-mediated angiogenesis was confirmed by a significant increase in microvessel's density in the penumbra area and an increase in von Willebrand factor (vWF) protein expression of the morin-treated rats. Moreover, the rotarod and pole tests also demonstrated morin increased functional recovery in the morin-treated rats compared to the vehicle rats. Therefore, our data exposed that morin promotes angiogenesis and improves functional outcomes in MCAO and reperfusion rats.

Dietary supplementation with L-arginine between days 14 and 25 of gestation enhances NO and polyamine syntheses and theexpression of angiogenic proteins in porcine placentae.

Dietary supplementation with 0.4 or 0.8% L-arginine (Arg) to gilts between days 14 and 25 of gestation enhances embryonic survival and vascular development in placentae; however, the underlying mechanisms are largely unknown. This study tested the hypothesis that Arg supplementation stimulated placental expression of mRNAs and proteins that enhance angiogenesis, including endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), placental growth factor (PGF), GTP cyclohydrolase-I (GTP-CH1), ornithine decarboxylase (ODC1), and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2). Beginning on the day of breeding, gilts were fed daily 2kg of a corn-soybean meal-based diet supplemented with 0.0 (control), 0.4, or 0.8% Arg. On day 25 of gestation, gilts were hysterectomized to obtain uteri and conceptuses for histochemical and biochemical analyses. eNOS and VEGFR1 proteins werelocalized to endothelial cells of maternal uterineblood vessels and to the uterine luminal epithelium, respectively. Compared with the control, dietary supplementation with 0.4 or 0.8% Arg increased (P < 0.05) the amounts of nitrite plus nitrate (NOx; oxidation products of NO) and polyamines in allantoic and amniotic fluids, concentrations of NOx, tetrahydrobiopterin (BH4, an essential cofactor for all NOS isoforms) and polyamines in placentae, as well as placental protein abundances of GTP-CH1 (the key enzyme for BH4 production) and ODC1 (the key enzyme for polyamine synthesis). Placental mRNA levelsfor GTP-CH1,eNOS, PGF,VEGF, and VEGFR2 increased in response to both 0.4% and 0.8% Arg supplementation. Collectively, these results indicate that dietary Arg supplementation to gilts between days 14 and 25 of pregnancy promotes placental angiogenesis by increasing the expression of mRNAs and proteins for angiogenic factors as well as NO andpolyaminesyntheses.

Therapeutic effect of a traditional Chinese medicine formulation on experimental choroidal neovascularization in mouse.

To investigate therapeutic effects of traditional Chinese medicine formulations, Hexuemingmu (HXMM) on laser-induced choroidal neovascularization (CNV) and follow-up effect in mice. C57BL/6 mice of 8-week-old were used and CNV was induced with 577 nm laser photocoagulation. Animals were randomly divided into groups and different doses of HXMM were administered daily. One, four, and eight weeks after the intervention, the electroretinogram (ERG), fundus fluorescence angiography, choroidal flat mount and immunofluorescence staining were preformed to evaluate the function and CNV formation. The expression levels of angiogenic proteins were determined by Western blotting and immunofluorescence staining. An analysis of variance and Kruskal-Wallis test were used to test the differences among the groups. The results showed that HXMM effectively increased amplitude of ERG of mice (P<0.05), alleviated fundus CNV leakage (P<0.05), and reduced the area of neovascularization and the expression of angiogenic proteins (P<0.05) after laser-induced CNV. HXMM can protect the retinal function of mice after laser-induced CNV, and inhibit the CNV development.

Intracellular Signaling Responses Induced by Radiation within an In Vitro Bone Metastasis Model after Pre-Treatment with an Estrone Analogue.

2-Ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) is an in silico-designed estradiol analogue which has improved the parent compound’s efficacy in anti-cancer studies. In this proof-of-concept study, the potential radiosensitizing effects of ESE-16 were investigated in an in vitro deconstructed bone metastasis model. Prostate (DU 145) and breast (MDA-MB-231) tumor cells, osteoblastic (MC3T3-E1) and osteoclastic (RAW 264.7) bone cells and human umbilical vein endothelial cells (HUVECs) were representative components of such a lesion. Cells were exposed to a low-dose ESE-16 for 24 hours prior to radiation at non-lethal doses to determine early signaling and molecular responses of this combination treatment. Tartrate-resistant acid phosphatase activity and actin ring formation were investigated in osteoclasts, while cell cycle progression, reactive oxygen species generation and angiogenic protein expression were investigated in HUVECs. Increased cytotoxicity was evident in tumor and endothelial cells while bone cells appeared to be spared. Increased mitotic indices were calculated, and evidence of increased deoxyribonucleic acid damage with retarded repair, together with reduced metastatic signaling was observed in tumor cells. RAW 264.7 macrophages retained their ability to differentiate into osteoclasts. Anti-angiogenic effects were observed in HUVECs, and expression of hypoxia-inducible factor 1-α was decreased. Through preferentially inducing tumor cell death and potentially inhibiting neovascularization whilst preserving bone physiology, this low-dose combination regimen warrants further investigation for its promising therapeutic application in bone metastases management, with the additional potential of limited treatment side effects.

Heat shock protein A12B gene therapy improves perfusion, promotes neovascularization, and decreases fibrosis in a murine model of hind limb ischemia

BackgroundHeat shock protein A12B expressed in endothelial cells is important and required for angiogenesis to form functional vessels in ischemic tissue. We have previously shown the cardioprotective effects of heat shock protein A12B overexpression in a rat model of diabetic myocardial infarction. In this study, we aim to explore the role of heat shock protein A12B in a surgically-induced murine hind-limb ischemia model. Materials and MethodsAdult 8- to 12-week-old C57BL/6J mice were divided into 2 groups: treated with Adeno.LacZ (control group) and with Adeno.HSPA12B (experimental group) and, with both groups subjected to right femoral artery ligation. Immediately after surgery, mice in both groups received either Adeno.HSPA12B or Adeno.LacZ (1 × 109 plaque forming units) in both the semimembranosus and gastrocnemius muscles of the right limb. The left limb served as the internal control. Both groups underwent serial laser Doppler imaging preoperatively, and again postoperatively until 28 days. Immunohistochemical analysis was performed 3 and 28 days post-surgery. ResultsMice in the Adeno.HSPA12B gene therapy group showed improved motor function and a significantly higher blood perfusion ratio on postoperative days 21 and 28, along with better motor function. Immunohistochemical analysis showed increased expression of vascular endothelial growth factor, thioredoxin-1, heme oxygenase, and hypoxia-inducible factor 1α, along with a decreased expression of A-kinase-anchoring protein 12 and thioredoxin-interacting protein levels. The Adeno.HSPA12B-treated group also showed increased capillary and arteriolar density and an increased capillary-myocyte ratio, along with reduced fibrosis compared to the Adeno.LacZ group. ConclusionOur study demonstrates that targeted Adeno.HSPA12B gene delivery into ischemic muscle enhances perfusion and angiogenic protein expression. This molecule shows promise for the management of peripheral vascular disease as a potential target for clinical trials and subsequent drug therapy.

328 Chorionic villi angiogenic protein expression in normal human pregnancy

Our goal was to examine the changes in chorionic villi (CV) expression of angiogenic proteins VEGF165, VEGF165b and MMP-9 throughout gestation in normal human pregnancy. Placentas were obtained from normal pregnant women who underwent elective abortion in the first and second trimesters of pregnancy, and from women who delivered at term. CV protein expressions were analyzed by ELISA using monoclonal antibodies to human VEGF165, VEGF165b and MMP-9 proteins (R&D Systems, Minneapolis, MN). Non-parametric test considered p<0.05 as significant. Our study highlights gestational age-specific alterations in all three CV proteins. VEGF165b protein peaked in the 2nd trimester while VEGF165 protein peaked in the third. MMP-9 protein progressively increased with increase in gestational age. Spearman's correlation showed that VEGF165, VEGF165b and MMP-9 proteins were significantly correlated with gestational age in days (r2=+0.236, p=0.001; r2= +0.158, p=0.028; r2=+0.475, p=0.0001, respectively). In the 3rd trimester, a significant positive correlation was seen between VEGF165 and VEGF165b (r2=+0.368, p=0.001); and a significant negative correlation was seen between VEGF165 and MMP-9 proteins (r2= -0.306, p=0.003). Of the three proteins selected, VEGF165 is the dominant angiogenic protein and its overexpression can result in tumorigenesis. VEGF165b is down-regulated in tumors. MMP-9 degrades ECM and promotes endothelial cell migration and is considered angiogenic. The 3rd trimester of human pregnancy is a unique phase when maximum angiogenesis needs to occur, yet as pregnancy approaches parturition the angiogenic process needs to be terminated. The correlation seen between the two isoforms of VEGF in the 3rd trimester suggest that when fetal demand is at its peak the two VEGF isoforms work synergistically to maximize angiogenesis. And, the ample presence of VEGF165b in the 3rd trimester and the inverse correlation seen between MMP-9 and VEGF165 could be mechanisms at place to restrict overexpression of VEGF165 and placental angiogenesis as pregnancy approaches parturition.

Local intramyocardial delivery of bioglass with alginate hydrogels for post-infarct myocardial regeneration

Heart failure (HF) is a common and serious manifestation after myocardial infarction (MI). Despite their clinical importance, current treatments for MI still have several limitations. Revascularization has been proven to have positive effects on MI-induced damage. Currently biomaterial-based angiogenesis strategies represent potential candidates for MI treatment. Bioglass (BG) is a commercially available family of bioactive glasses. BG has angiogenic properties and thus might be an attractive alternative for MI treatments. Here, we loaded BG in sodium alginate (BGSA), locally injected it into peri-infarct myocardial tissue and examined its suitability for inducing cardiac angiogenesis and eventually improving cardiac function following MI. Cardiac function was evaluated via echocardiography. Infarct morphometry, angiogenesis, apoptosis and angiogenic protein expression were all analysed 4 weeks after BGSA injection. Compared with the control treatment, BGSA was sufficient to prompt angiogenesis, suppress apoptosis, up-regulate the expression of angiogenic proteins, attenuate infarct size, preserve wall thickness and eventually improve cardiac function. Our results demonstrate the feasibility and effectiveness of BGSA in myocardial regeneration via angiogenesis, suggesting that BGSA is a potential therapeutic strategy for post-infarct myocardial regeneration.

Expression of Angiogenic Proteins in Tumor and Stroma Affects Survival in Patients With Gastric Cancer

BackgroundGastric cancer is one of the most frequent malignancies worldwide. Angiogenic growth factors play a crucial role in mediating the crosstalk between cancer cells and the surrounding microenvironment. In this exploratory study, we investigate the impact of angiogenic proteins within the tumor cell or stroma compartment on survival of patients with gastric cancer. Materials and methodsIn 29 patients, tumor and stromal compartments were separated using laser capture microdissection. Angiogenic protein expression was measured using a bead-based immunoassay and correlated with tumor stage and overall survival. ResultsOverall survival was significantly shorter in patients with a high stroma concentration of vascular endothelial growth factor (VEGF)-A (23.5 (±17.6) versus 33.6 (±21.0) mo; P = 0.009) and stem cell factor (22.2 (±18.5) versus 33.6 (±21.8) mo; P = 0.01) compared with patients with a low stroma concentration. High stromal VEGF-D showed a trend toward worse survival (26.8 (±22.0) versus 37.2 (±19.0) mo; P = 0.09). We did not observe any significant correlation between tumor-specific expression of angiogenic cytokines and survival. ConclusionsThis translational study highlights the difference in clinical impact between tumor and stromal expression of angiogenic proteins. Compartment-specific concentrations of VEGF-A and stem cell factor affect the clinical prognosis and help to identify the best therapy for patients with gastric cancer.

N-Ethyl-n-Nitrosourea Induced Leukaemia in a Mouse Model through Upregulation of Vascular Endothelial Growth Factor and Evading Apoptosis.

Chemical carcinogens are commonly used to investigate the biology and prognoses of various cancers. This study investigated the mechanism of leukaemogenic effects of n-ethyl-n-nitrosourea (ENU) in a mouse model. A total of 14 3-week-old male Institute of Cancer Research (ICR)-mice were used for the study. The mice were divided into groups A and B with seven mice each. Group A served as the control while group B received intraperitoneal (IP) injections of 80 mg/kg ENU twice with a one-week interval and were monitored monthly for 3 months for the development of leukaemia via blood smear examination. The mice were sacrificed humanely using a CO2 chamber. Blood, spleen, lymph nodes, liver, kidney and lung samples were collected for blood smear examination and histopathological evaluation. The expression of angiogenic protein (VEGF), and pro and anti-apoptotic proteins (BCL2 and BAX), was detected and quantified using Western blot technique. Leukaemia was confirmed by the presence of numerous blast cells in the peripheral blood smear in group B. Similarly, the VEGF and BCL2 proteins were significantly (p < 0.05) upregulated in group B compared to A. It is concluded that IP administration of 80 mg/kg ENU induced leukaemia in ICR-mice 12 weeks post administration through upregulation of angiogenic and anti-apoptotic proteins: VEGF and BCL2.