Impact of High Sodium Diet on Neovascularization and Osseointegration around Titanium Implant:An in Vivo and in Vitro Study

Objective A high sodium(HS)diet is believed to affect bone metabolism processes.Clarifying its impact on osseointegration of titanium(Ti)implants holds significant implications for postoperative dietary management of implanted patients.Methods This investigation probed the impact of sodium ions(Na^(+))on neovascularization and osteogenesis around Ti implants in vivo,utilizing micro-computed tomography,hematoxylin and eosin staining,and immunohistochemical analyses.Concurrently,in vitro experiments assessed the effects of varied Na^(+)concentrations and exposure durations on human umbilical vein endothelial cells(HUVECs)and MC3T3-E1 cells.Results In vivo,increased dietary sodium(0.8%-6.0%)led to a substantial decline in CD34 positive HUVECs and new bone formation around Ti implants,alongside an increase in inflammatory cells.In vitro,an increase in Na^(+)concentration(140-150 mmol/L)adversely affected the proliferation,angiogenesis,and migration of HUVECs,especially with prolonged exposure.While MC3T3-E1 cells initially exhibited less susceptibility to high Na^(+)concentrations compared to HUVECs during short-term exposure,prolonged exposure to a HS environment progressively diminished their proliferation,differentiation,and osteogenic capabilities.Conclusion These findings suggest that HS diet had a negative effect on the early osseointegration of Ti implants by interfering with the process of postoperative vascularized bone regeneration.

Silk-based nerve guidance conduits with macroscopic holes modulate the vascularization of regenerating rat sciatic nerve

Peripheral nerve injuries induce a severe motor and sensory deficit. Since the availability of autologous nerve transplants for nerve repair is very limited, alternative treatment strategies are sought, including the use of tubular nerve guidance conduits(tNGCs). However, the use of tNGCs results in poor functional recovery and central necrosis of the regenerating tissue, which limits their application to short nerve lesion defects(typically shorter than 3 cm). Given the importance of vascularization in nerve regeneration, we hypothesized that enabling the growth of blood vessels from the surrounding tissue into the regenerating nerve within the tNGC would help eliminate necrotic processes and lead to improved regeneration. In this study, we reported the application of macroscopic holes into the tubular walls of silk-based tNGCs and compared the various features of these improved silk^(+) tNGCs with the tubes without holes(silk^(–) tNGCs) and autologous nerve transplants in an 8-mm sciatic nerve defect in rats. Using a combination of micro-computed tomography and histological analyses, we were able to prove that the use of silk^(+) tNGCs induced the growth of blood vessels from the adjacent tissue to the intraluminal neovascular formation. A significantly higher number of blood vessels in the silk^(+) group was found compared with autologous nerve transplants and silk^(–), accompanied by improved axon regeneration at the distal coaptation point compared with the silk^(–) tNGCs at 7 weeks postoperatively. In the 15-mm(critical size) sciatic nerve defect model, we again observed a distinct ingrowth of blood vessels through the tubular walls of silk^(+) tNGCs, but without improved functional recovery at 12 weeks postoperatively. Our data proves that macroporous tNGCs increase the vascular supply of regenerating nerves and facilitate improved axonal regeneration in a short-defect model but not in a critical-size defect model. This study suggests that further optimization of the macroscopic holes silk^(+) tNGC approach containing macroscopic holes might result in improved grafting technology suitable for future clinical use.

Temporal and spatial regulation of biomimetic vascularization in 3D-printed skeletal muscles

In the intricate skeletal muscle tissue,the symbiotic relationship between myotubes and their supporting vasculature is pivotal in delivering essential oxygen and nutrients.This study explored the complex interplay between skeletal muscle and endothelial cells in the vascularization ofmuscle tissue.By harnessing the capabilities of three-dimensional(3D)bioprinting and modeling,we developed a novel approach involving the co-construction of endothelial and muscle cells,followed by their subsequent differentiation.Our findings highlight the importance of the interaction dynamics between these two cell types.Notably,introducing endothelial cells during the advanced phases of muscle differentiation enhanced myotube assembly.Moreover,it stimulated the development of the vascular network,paving the way for the early stages of vascularized skeletal muscle development.The methodology proposed in this study indicates the potential for constructing large-scale,physiologically aligned skeletal muscle.Additionally,it highlights the need for exploring the delicate equilibrium and mutual interactions between muscle and endothelial cells.Based on the multicell-type interaction model,we can predict promising pathways for constructing even more intricate tissues or organs.

Salvage anastomosis in free PAP-flap breast reconstruction:What about free flap neovascularization?

Since the emergence of microsurgery in reconstructive surgery, free flaps have become a key tool in the management of patients with breast cancer. One such flap is the profunda artery perforator(PAP) flap. To date,there is no scientific consensus on whether voluminous free flaps remain dependent on their vascular pedicle throughout their lifespan. Therefore, the pedicle should always be carefully protected during revision surgery.In this article, we review the case of a middle-aged woman who suffered a pedicle transection needing reanastomosis during revision surgery six months after free-flap breast reconstruction. A 52-year-old woman who noticed a firm nodule in her right breast and armpit was referred to our department for surgical management. The Caucasian woman presented with no significant medical history or symptoms at the first consultation. Ultrasound-guided biopsy confirmed an invasive grade Ⅲ lobular carcinoma. Following staging,the patient underwent neoadjuvant chemotherapy before a right mastectomy with a complete homolateral axillary lymph node dissection and postoperative radiotherapy. One year after completing radiotherapy, free flap reconstruction with a PAP flap was performed, and six months later, revision surgery was required to enhance the volume of the reconstructed breast with a tissue expander and later an implant. Unfortunately,pedicle transection occurred during revision surgery, causing complete devascularization of the flap, which was confirmed by intraoperative Indocyanine Green imaging. The authors elected to perform salvage reanastomosis during the surgery. In keeping with the author’s 23-year experience with free flaps, the vascular pedicle should always be preserved in voluminous free flaps, as neovascularization alone may not ensure whole flap survival. The authors suggest always attempting re-anastomosis if vessels are compromised during revision surgery.

The role of vascularization in nerve regeneration of nerve graft

Vascularization is an important factor in nerve graft survival and function. The specific molecular regulations and patterns of angiogenesis following peripheral nerve injury are in a broad complex of pathways. This review aims to summarize current knowledge on the role of vascularization in nerve regeneration, including the key regulation molecules, and mechanisms and patterns of revascularization after nerve injury. Angiogenesis, the maturation of pre-existing vessels into new areas, is stimulated through angiogenic factors such as vascular endothelial growth factor and precedes the repair of damaged nerves. Vascular endothelial growth factor administration to nerves has demonstrated to increase revascularization after injury in basic science research. In the clinical setting, vascularized nerve grafts could be used in the reconstruction of large segmental peripheral nerve injuries. Vascularized nerve grafts are postulated to accelerate revascularization and enhance nerve regeneration by providing an optimal nutritional environment, especially in scarred beds, and decrease fibroblast infiltration. This could improve functional recovery after nerve grafting, however, conclusive evidence of the superiority of vascularized nerve grafts is lacking in human studies. A well-designed randomized controlled trial comparing vascularized nerve grafts to non-vascularized nerve grafts involving patients with similar injuries, nerve graft repair and follow-up times is necessary to demonstrate the efficacy of vascularized nerve grafts. Due to technical challenges, composite transfer of a nerve graft along with its adipose tissue has been proposed to provide a healthy tissue bed. Basic science research has shown that a vascularized fascial flap containing adipose tissue and a vascular bundle improves revascularization through excreted angiogenic factors, provided by the stem cells in the adipose tissue as well as by the blood supply and environmental support. While it was previously believed that revascularization occurred from both nerve ends, recent studies propose that revascularization occurs primarily from the proximal nerve coaptation. Fascial flaps or vascularized nerve grafts have limited applicability and future directions could lead towards off-the-shelf alternatives to autografting, such as biodegradable nerve scaffolds which include capillary-like networks to enable vascularization and avoid graft necrosis and ischemia.

Adenomyosis uterine innervation in mice correlates to nerve growth factor expression,inflammation,and vascularization

BACKGROUND： Studies have shown that abnormal innervation is an important factor impacting occurrence and development of pathological pain in endometriosis. OBJECTIVE： To observe uterine innervation of adenomyosis mice and to analyze the cause of innervation changes due to nerve growth factor （NGF） expression, inflammation, and vascularization. DESIGN, TIME AND SETTING： This randomized, controlled, animal experiment was performed at the Research Institute of Obstetrics and Gynecology Hospital, and Central Laboratory of Zhongshan Hospital, Fudan University from March to December 2008. MATERIALS： Tamoxifen was provided by Fudan Forward, China. Rabbit anti-mouse NGF was purchased from Santa Cruz Corporation, USA; rabbit anti-protein gene product 9.5 （PGP9.5） and rabbit anti-substance P （SP） were purchased from Chemicon, USA. METHODS： A total of 40 newborn ICR mice were randomly assigned to adenomyosis model and control groups, with 20 animals in each group. Mice in the adenomyosis model group were orally administrated 2.7 μmol/kg tamoxifen on days 2-5 after birth, while the controls were not treated. MAIN OUTCOME MEASURES： Both uteri from all mice were harvested at days 135-145 after birth Expressions of polyclonal PGP9.5 and SP were immunohistochemically detected to demonstrate pan- and sensory nerve fibers. Microvessel density was quantified in the endometrium and myometrium using immunochemical staining for polyclonal rabbit anti-CD31, which stained vessels. Gene expression for NGF, high-affinity tyrosine kinase receptor （trkA）, p75 neuretrophin receptor （p75NTR）, bradykinin receptor-1 （BKR-1）, and 2 （BKR-2）, as well as substance P receptor （neurokininl receptor, NK1-R）, were detected by reverse transcription-polymerase chain reaction. NGF-13 protein expression was detected by Western blot analysis. RESULTS： More nerve fibers were stained with PGP9.5 in the endometrium and myometrium, and with SP in the endometrium, in adenomyosis mice compared with controls （P 〈 0.01 and P 〈 0.05）. Microvessel density in the myometrium of adenomyosis mice was significantly greater than the controls （P 〈 0.01）. In the uterus of adenomyosis mice, mRNA expression of NGF and its two receptors （trkA and p75 NTR）, BKR-1, and NK1-R, as well as protein expression of NGF-β, were greater than the control mice （P 〈 0.01 or P 〈 0.05）. CONCLUSION： Uterine innervation in the adenomyosis mice was increased compared with the controls. Moreover, NGF expression, inflammation, and vascularization, which have been shown to be impact factors of innervation, were abnormal in the uteri of adenomyosis mice.

Effects of endostatin on expression of vascular endothelial growth factor and its receptors and neovascularization in colonic carcinoma implanted in nude mice

AIM:To investigate the antiangiogenic effects of endostatin on colonic carcinoma cell line implanted in nude mice and its mechanism. METHODS:Nude mice underwent subcutaneous injection with LS-174t colonic carcinoma cell line to generate carcinoma and were randomly separated into two groups.Mice received injection of vehicle or endostatin every day for two weeks. After the tumor was harvested,the tumor volumes were determined,and the expressions of CD34,VEGF and FIk-1 were examined by immunohistochemical method. RESULTS:Tumor volume was significantly inhibited in the endostatin group(84.17%)and tumor weight was significantly inhibited in the endostatin group(0.197±0.049) compared to the control group(1.198±0.105)(F=22.56, P=0.001),microvessel density(MVD)was significantly decreased in the treated group(31.857±3.515)compared to the control group(100.143±4.290)(F=151.62,P<0.001). Furthermore,the expression of FIk-1 was significantly inhibited in the treated group(34.29%) ompared to the control group(8.57%)(X^2=13.745,P=0.001).However no significant decrease was observed in the expression of vascular endothelial growth factor(VEGF)between these two groups(X^2=0.119,P=0.730). CONCLUSION:Endostatin can inhibit tumor growth and angiogenesis by blocking Vegf/FIk-1 pathway.This experiment provides the theory basis for developing a new anti-carcinoma drug through studying the properties of anti-angiogenesis inhibitors.

Effects of long non-coding RNA myocardial infarction-associated transcript on retinal neovascularization in a newborn mouse model of oxygen-induced retinopathy

Whether long non-coding RNA myocardial infarction-associated transcript is involved in oxygen-induced retinopathy remains poorly understood. To validate this hypothesis, we established a newborn mouse model of oxygen-induced retinopathy by feeding in an oxygen concentration of 75 ± 2% from postnatal day 8 to postnatal day 12, followed by in normal air. On postnatal day 11, the mice were injected with the myocardial infarction-associated transcript siRNA plasmid via the vitreous cavity to knockdown long non-coding RNA myocardial infarction-associated transcript. Myocardial infarction-associated transcript siRNA transcription significantly inhibited myocardial infarctionassociated transcript mRNA expression, reduced the phosphatidylinosital-3-kinase, phosphorylated Akt and vascular endothelial growth factor immunopositivities, protein and mRNA expression, and alleviated the pathological damage to the retina of oxygen-induced retinopathy mouse models. These findings suggest that myocardial infarction-associated transcript is likely involved in the retinal neovascularization in retinopathy of prematurity and that inhibition of myocardial infarction-associated transcript can downregulate phosphatidylinosital-3-kinase, phosphorylated Akt and vascular endothelial growth factor expression levels and inhibit neovascularization. This study was approved by the Animal Ethics Committee of Shengjing Hospital of China Medical University, China(approval No. 2016 PS074 K) on February 25, 2016.

Impact of Lycium Barbarum Polysaccharide and Danshensu on vascular endothelial growth factor in the process of retinal neovascularization of rabbit

AIM:To discuss the impact of Lycium Barbarum Polysaccharide (LBP) and Danshensu purified from Traditional Chinese Medicine (TCM) on vascular endothelial growth factor (VEGF) of rabbits with retinal neovascularization. METHODS:Forty rabbits were divided into normal control group, model control group, LBP group and Danshensu group. Animals in the normal control group were fed in the normal oxygen environment. Animals in the other three groups were put into the environment with 70% oxygen for 5 days in order to build the model of oxygen-induced vascular proliferation retinopathy. And then different TCM extract was injected into the abdominal cavities of these annimals. After 7 days, the VEGF content of in the serum of rabbit was measured by double antibody sandwich method. RESULTS:Data analysis indicated that VEGF content was as follows:Danshensu group was lower than model control group (12.92 ±3.84ng/L vs 19.32 ±4.15ng/L, P 【 0.05); LBP group and normal control group were lower than model control group (12.92±3.84ng/L, 9.26±1.61ng/L vs 19.32±4.15ng/L, P【0.01); total blood viscosity, plasma viscosity, cholesterol content, fibrinogen content and triacylglycerol content after peritoneal injection of LBP and Danshensu were obviously lower than before injection. CONCLUSION:TCM extract-LBP and Danshensu can prominently reduce the content of VEGF in the process of vascular proliferative retinopathy of rabbit; can prevent the occurrence of retinal microvascular disease by improving partial oxygen -deficient environment or affecting all kinds of new growth factor.

Elevation of vascular endothelial growth factor production and its effect on revascularization and function of graft islets in diabetic rats

AIM: To determine whether the elevated vascular endothelial growth factor (VEGF) expression produced by the transfected vascular endothelial cells (VECs) could stimulate angiogenesis of the graft islets and exert its effect on the graft function. METHODS: Thirty diabetic recipient rats were divided into three groups (n = 10 per group). In the control group,300 IEQ islets were transplanted in each rat under the capsule of the right kidney,which were considered as marginal grafts. In the VEC group,VEC together with the islets were transplanted in each rat. In the VEGF group,VEC transfected by pIRES2-EGFP/ VEGF165 plasmid and the islets were transplanted in each rat. Blood glucose and insulin levels were evaluated every other day after operation. Intravenous glucose tolerance test (IVGTT) was performed 10 d after the transplantation. Hematoxylin and eosin (HE) staining was used to evaluate the histological features of the graft islets. Immunohistochemical staining was used to detect insulin-6,VEGF and CD34 (MVD) expression in the graft islets. RESULTS: Blood glucose and insulin levels in the VEGF group restored to normal 3 d after transplantation. In contrast,diabetic rats receiving the same islets with or without normal VECs displayed moderate hyperglycemia and insulin,without a significant difference between these two groups. IVGTT showed that both the amplitude of blood glucose induction and the kinetics of blood glucose in the VEGF group restored to normal after transplantation. H&E and immunohistochemical staining showed the presence of a large amount of graft islets under the capsule of the kidney,which were positively stained with insulin-6 and VEGF antibodies in the VEGF group. In the cell masses,CD34-stained VECs were observed. The similar masses were also seen in the other two groups,but with a fewer positive cells stained with insulin-6 and CD34 antibodies. No VEGF-positive cells appeared in these groups. Microvessel density (MVD) was significantly higher in the VEGF group compared to the other two groups. CONCLUSION: Elevated VEGF production by trans-fected vascular endothelial cells in the site of islet transplantation stimulates angiogenesis of the islet grafts. The accelerated islet revascularization in early stage could improve the outcome of islet transplantation,and enhance the graft survival.

Inhibition of LY294002 in retinal neovascularization via down-regulation the PI3K/AKT-VEGF pathway in vivo and in vitro

AIM： To investigate the effects of the phosphatidylinositol 3-kinase（PI3 K） inhibitor LY294002 on retinal neovascularization（RNV） in the oxygen-induced retinopathy（OIR） mouse model and human umbilical vein endothelial cells（HUVECs）.METHODS： C57 BL/6 J mice were randomly divided into normoxia-control, OIR-control and LY294002 treatment groups. LY294002 or phosphate-buffered solution was intraperitoneally injected daily into mouse pups from P6 to P9 in LY294002 treatment group or OIR-control group. Morphological and pathological changes in RNV, as well as expression levels of PI3 K, serine-threonine kinase（AKT） and vascular endothelial growth factor（VEGF） were observed. HUVECs treating with LY294002 were exposed to hypoxia; the expression of PI3 K, AKT and VEGF were examined by Western blot and RT-PCR analyses.RESULTS： Compared with the OIR-control group, LY294002 significantly inhibit RNV. Adenosine diphosphatase（ADPase） staining and hematoxylin and eosin staining indicated that the clock hour scores of neovascularization and the nuclei of pre-retinal neovascular cells in the LY294002 treatment group were clearly less than those in the OIR-control group（1.41±0.52 vs 6.20±1.21; 10.50±1.58 vs 22.25±1.82, both P〈0.05）. Intravitreal injection of LY294002（in the LY294002 treatment group） markedly decreased PI3 K/AKT-VEGF expression compared with the OIR-control group by immunohistochemistry, Western blotting and RT-PCR（all P〈0.05）. In HUVECs treated with hypoxia, expression of PI3 K, AKT and VEGF were downregulated in the hypoxia-LY294002 group（all P〈0.05）.CONCLUSION： The PI3 K inhibitor LY294002 can inhibit RNV by downregulating PI3 K, AKT, and VEGF expression in vivo and in vitro. LY294002 may provide an effective method for preventing retinopathy of prematurity（ROP）.

Inhibition of choroidal neovascularization by lentivirusmediated PEDF gene transfer in rats

AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.

Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

BACKGROUND A major problem in the healing of bone defects is insufficient or absent blood supply within the defect.To overcome this challenging problem,a plethora of approaches within bone tissue engineering have been developed recently.Bearing in mind that the interplay of various diffusible factors released by endothelial cells(ECs)and osteoblasts(OBs)have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions,we set the focus on the simultaneous application of these cell types together with platelet-rich plasma(PRP)as a growth factor reservoir within ectopic bone tissue engineering constructs.AIM To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells(ASCs)induced into ECs and OBs.METHODS ASCs isolated from adipose tissue,induced in vitro into ECs,OBs or just expanded were used for implant construction as followed:BPEO,endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix;BPUI,uninduced ASCs with PRP and bone mineral matrix;BC(control),only bone mineral matrix.At 1,2,4 and 8 wk after subcutaneous implantation in mice,implants were extracted and endothelial-related and bone-related gene expression were analyzed,while histological analyses were performed after 2 and 8 wk.RESULTS The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation.BC had the lowest endothelial-related gene expression,weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO.Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO.BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs.Except Vwf,endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk.Tissue regression was noticed in BPEO constructs after 8 wk.CONCLUSION Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis,but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.

Penile revascularization--contemporary update

Contemporary therapies for erectile dysfunction are generally targeted towards older men and universally engage pharmacological and/ or device related treatment options. Penile revascularization, using microvascular arterial bypass surgical techniques, is a non-pharmacological, non-device-related, and reconstructive surgical strategy for men with erectile dysfunction that was first described by Dr Vaclav Michal in 1973.

The process of retinal vascularization in retinopathy of prematurity after ranibizumab treatment in China

AIM: To explore the process of retinal vascularization and risk factors for retinopathy of prematurity(ROP) treated with intravitreal ranibizumab(IVR) as monotherapy.METHODS: Infants with type 1 ROP who received IVR as primary treatment from August 2014 to October 2016 at Peking University People’s Hospital’s Ophthalmology Department were included in the study. All eyes received 0.25 mg ranibizumab at initial treatment. Retinal vascularization was evaluated clinically. Potential risk factors were also recorded and examined.RESULTS: Retinal vascularization was completed in 126 eyes(62.7%), and retinal vascularization terminated in zone II and zone III with 16 eyes(7.9%) and 44 eyes(21.9%), respectively, after more than 1-year follow-up. In multivariate regression analysis, lower birth weight(BW), severity of ROP and repeated injections were found to be risk factors for peripheral avascular area(P<0.05). CONCLUSION: In our retrospective study, 29.8% of the ROP eyes treated with ranibizumab have peripheral avascular area at the last follow-up. Lighter BW and the severity of ROP are risk factors. Furthermore, repeated injections also increase the risk of retinal peripheral avascular area remaining in ROP patients.

Anti-apoptosis effects of vascular endothelial cadherin in experimental corneal neovascularization

AIMTo explore the effects and mechanism of vascular endothelial cadherin (VE-cadherin) on experimental corneal neovascularization (CRNV).METHODSMouse corneas were burned with sodium hydroxide to build a CRNV model. The burned corneas were locally administrated with anti-mouse VE-cadherin neutralizing antibody. Annexin V and cluster of differentiation 31 (CD31) double staining was used to measure vascular endothelial cell apoptosis with the use of flow cytometry (FCM). The protein expression of NADPH oxidase 2 (Nox2), caspase-3, and protein kinase C (PKC) in the burned corneas were examined by Western blot. Human retinal endothelial cell (HREC) proliferation was detected using a Cell Counting Kit 8 (CCK-8) assay in vitro.RESULTSThe amount of CRNV peaked two weeks after the alkali burn. FCM confirmed that VE-cadherin neutralizing antibody treatment increased CD31 positive cell apoptosis. Western blot revealed that the intracorneal protein expression of Nox2 and caspase-3 were up-regulated, while PKC was down-regulated in the VE-cadherin neutralizing antibody administrated group. CCK-8 assay showed that VE-cadherin neutralizing antibody markedly inhibited HREC proliferation.CONCLUSIONVE-cadherin exhibited an anti-apoptosis effect through enhanced PKC signaling and an enhanced cell proliferation pathway.

MicroRNAs in laser-induced choroidal neovascularization in mice and rats:their expression and potential therapeutic targets

Choroidal neovascularization characterizes wet age-related macular degeneration.Choroidal neovascularization formation involves a primarily angiogenic process that is combined with both inflammation and proteolysis.A primary cause of choroidal neovascularization pathogenesis is alterations in pro-and anti-angiogenic factors derived from the retinal pigment epithelium,with vascular endothelium growth factor being mainly responsible for both clinical and experimental choroidal neovascularization.MicroRNAs(miRNAs)which are short,non-coding,endogenous RNA molecules have a major role in regulating various pathological processes,including inflammation and angiogenesis.A review of recent studies with the mouse laser-induced choroidal neovascularization model has shown alterations in miRNA expression in choroidal neovascularization tissues and could be potential therapeutic targets for wet age-related macular degeneration.Upregulation of miR-505(days 1 and 3 post-laser),miR-155(day 14)occurred in retina;miR-342-5p(days 3 and 7),miR-126-3p(day 14)in choroid;miR-23a,miR-24,miR-27a(day 7)in retina/choroid;miR-505(days 1 and 3)in retinal pigment epithelium/choroid;downregulation of miR-155(days 1 and 3),miR-29a,miR-29b,miR-29c(day 5),miR-93(day 14),miR-126(day 14)occurred in retinal pigment epithelium/choroid.Therapies using miRNA mimics or inhibitors were found to decrease choroidal neovascularization lesions.Choroidal neovascularization development was reduced by overexpression of miR-155,miR-188-5p,miR-(5,B,7),miR-126-3p,miR-342-5p,miR-93,miR-126,miR-195a-3p,miR-24,miR-21,miR-31,miR-150,and miR-184,or suppression of miR-505,miR-126-3p,miR-155,and miR-23/27.Further studies are warranted to determine miRNA expression in mouse laser-induced choroidal neovascularization models in order to validate and extend the reported findings.Important experimental variables need to be standardized;these include the strain and age of animals,gender,number and position of laser burns to the eye,laser parameters to induce choroidal neovascularization lesions including wavelength,power,spot size,and duration.

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.

Matrix metalloproteinase-9 and vascular endothelial growth factor expression change in experimental retinal neovascularization

AIM： To investigate the signal transduction mechanism of matrix metalloproteinase-9 （MMP-9） mediatedvascular endothelial growth factor （VEGF） expression and retinal neovascularization （RNV） in oxygen-induced retinopathy （OIR） model. METHODS： C57BL/6J mice were divided into four groups： control group, OIR group, OIR control group （phosphatebuffered saline by intravitreal injection） and treated group [tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） by intravitreal injection]. OIR model was established in C57BIJ6J mice exposed to 75% ＋2% oxygen for 5d. mRNA level and protein expression of MMP-9, TIMP-1 and VEGF were measured by real-time polymerase chain reaction and Western blotting, and located by immunohistochemistry. RESULTS： Levels of MMP-9 and VEGF in retina were significantly increased in animals with OIR and OIR control group. Levels of TIMP -1 in retina was significantly reduced in animals with OIR and OIR control group. Furthermore, a significant correlation was found between MMP-9 and VEGF. Intravitreal injection of TIMP- 1 significantly reduced MMP-9 and VEGF expression of the OIR mouse model （all P〈0.05）. CONCLUSION： These results demonstrate that MMP- 9-mediated up-regulation of VEGF promotes RNV in retinopathy of prematurity （ROP）. TIMP-1 may be a potential target for the prevention and treatment of ROP.

Mechanisms of vascularization in murine models of primary and metastatic tumor growth

Directed capillary ingrowth has long been considered synonymous with tumor vascularization.However,the vasculature of primary tumors and metastases is not necessarily formed by endothelial cell sprouting;instead,malignant tumors can acquire blood vessels via alternative vascularization mechanisms,such as intussusceptive microvascular growth,vessel co-option,and glomeruloid angiogenesis.Importantly,in response to anti-angiogenic therapies,malignant tumors can switch from one vascularization mechanism to another.In this article,we briefly review the biological features of these mechanisms and discuss on their significance in medical oncology.