VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel^?-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A₁₆₅ in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A₁₆₅ induced ~110 % increase in cell proliferation relative to basal conditions (～51 % without heparin). 2.62 pM VEGF-A₁₆₅ induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A₁₆₅ induced ～1.3-fold increased average rate of endothelial wound healing 4–18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A₁₆₅ was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A₁₆₅, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A₁₆₅ may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.     

Discovery of novel inhibitors of vascular endothelial growth factor-A–Neuropilin-1 interaction by structure-based virtual screening

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A₁₆₅ to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A₁₆₅ binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A₁₆₅ binding to recombinant NRP-1 with K_i of about 10 μM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.     

Carbohydrate-based peptidomimetics targeting neuropilin-1: Synthesis,molecular docking study and in vitro biological activities

Neuropilin-1 (NRP-1), a transmembrane glycoprotein acting as a co-receptor of VEGF-A, is expressed by cancer and angiogenic endothelial cells and is involved in the angiogenesis process. Taking advantage of functionalities and stereodiversities of sugar derivatives, the design and the synthesis of carbohydrate based peptidomimetics are here described. One of these compounds (56) demonstrated inhibition of VEGF-A₁₆₅ binding to NRP-1 (IC₅₀ = 39 μM) and specificity for NRP-1 over VEGF-R2. Biological evaluations were performed on human umbilical vein endothelial cells (HUVECs) through activation of downstream proteins (AKT and ERK phosphorylation), viability/proliferation assays and in vitro measurements of anti-angiogenic abilities.     

Human leptin induces angiogenesis in vivo

Leptin is an adipocyte-produced peptide, which plays a crucial role in the regulation of body weight. There is also evidence that leptin stimulates endothelial cell proliferation and the formation of capillary-like tubes in vitro. The disc angiogenesis system was used to measure the angiogenic effect of leptin in vivo. Discs containing 25, 50, 100 and 250 ng/ml of leptin were implanted subcutaneously in Wistar rats, removed after a growth period of 7 and 14 days, and compared with spontaneous growth controls and with positive controls containing equivalent doses of vascular endothelial growth factor (VEGF). Discs were examined morphologically for stroma and vessel development and by immunohistochemistry for quantitative evaluation of angiogenesis. The specificity of the angiogenic effect of leptin was tested by blocking leptin with a polyclonal anti-leptin antibody. Leptin induced a significant level of angiogenesis in a dose-dependent manner both at 7 and 14 days, with a peak at the dose of 100 ng/ml. The angiogenic activity of leptin was completely abolished by the anti-leptin neutralizing antibody. VEGF also induced significant dose-dependent angiogenesis at the same time points with a peak observed at a concentration of 100 ng/ml. The angiogenic response to leptin was significantly higher at 7 days compared with VEGF but not at the 14-day time point. In conclusion, leptin has a specific angiogenic effect in vivo, which is dose- and time-dependent in a concentration range of 25–250 ng/ml. This effect is equivalent to the angiogenic effect of VEGF but is evident earlier compared with VEGF.     

Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells

Recently, we identified a new Vascular Endothelial Growth Factor (VEGF)-A₁₆₅-induced gene Phactr-1, (Phosphatase Actin Regulator-1). We reported that Phactr-1 gene silencing inhibited tube formation in human umbilical endothelial cells (HUVECs) indicating a key role for Phactr-1 in tubulogenesis in vitro. In this study, we investigated the role of Phactr-1 in several cellular processes related to angiogenesis. We found that neuropilin-1 (NRP-1) and VEGF-R1 depletion inhibited Phactr-1 mRNA expression while NRP-2 and VEGF-R2 depletion had no effect. We described a new interaction site of VEGF-A₁₆₅ to VEGF-R1 in peptides encoded by exons 7 and 8 of VEGF-A₁₆₅. The specific inhibition of VEGF-A₁₆₅ binding on NRP-1 and VEGF-R1 by ERTCRC and CDKPRR peptides decreased the Phactr-1 mRNA levels in HUVECs indicating that VEGF-A₁₆₅-dependent regulation of Phactr-1 expression required both NRP-1 and VEGF-R1 receptors. In addition, upon VEGFA₁₆₅-stimulation Phactr-1 promotes formation and maintenance of cellular tubes through NRP-1 and VEGFR1. Phactr-1 was previously identified as protein phosphatase 1 (PP1) α-interacting protein that possesses actin-binding domains. We showed that Phactr-1 depletion decreased PP1 activity, disrupted the fine-tuning of actin polymerization and impaired lamellipodial dynamics. Taken together our results strongly suggest that Phactr-1 is a key component in the angiogenic process.     

Differential actions of VEGF-A isoforms on perichondrial angiogenesis during endochondral bone formation

During endochondral bone formation, vascular invasion initiates the replacement of avascular cartilage by bone. We demonstrate herein that the cartilage-specific overexpression of VEGF-A₁₆₄ in mice results in the hypervascularization of soft connective tissues away from cartilage. Unexpectedly, perichondrial tissue remained avascular in addition to cartilage. Hypervascularization of tissues similarly occurred when various VEGF-A isoforms were overexpressed in the chick forelimb, but also in this case perichondrial tissue and cartilage were completely devoid of vasculature. However, following bony collar formation, anti-angiogenic properties in perichondrial tissue were lost and perichondrial angiogenesis was accelerated by VEGF-A₁₄₆, VEGF-A₁₆₆, or VEGF-A₁₉₀. Once the perichondrium was vascularized, osteoclast precursors were recruited from the circulation and the induction of MMP9 and MMP13 can be observed in parallel with the activation of TGF-β signaling. Neither perichondrial angiogenesis nor the subsequent cartilage vascularization was found to be accelerated by the non-heparin-binding VEGF-A₁₂₂ or by the VEGF-A₁₆₆ΔE₁₆₂-R₁₆₆ mutant lacking a neuropilin-binding motif. Hence, perichondrial angiogenesis is a prerequisite for subsequent cartilage vascularization and is differentially regulated by VEGF-A isoforms.     

The acute phase reactant orosomucoid-1 is a bimodal regulator of angiogenesis with time- and context-dependent inhibitory and stimulatory properties

Background

Tissues respond to injury by releasing acute phase reaction (APR) proteins which regulate inflammation and angiogenesis. Among the genes upregulated in wounded tissues are tumor necrosis factor-alpha (TNFα) and the acute phase reactant orosomucoid-1 (ORM1). ORM1 has been shown to modulate the response of immune cells to TNFα, but its role on injury- and TNFα-induced angiogenesis has not been investigated. This study was designed to characterize the role of ORM1 in the angiogenic response to injury and TNFα.

Methods and Results

Angiogenesis was studied with in vitro, ex vivo, and in vivo angiogenesis assays. Injured rat aortic rings cultured in collagen gels produced an angiogenic response driven by macrophage-derived TNFα. Microarray analysis and qRT-PCR showed that TNFα and ORM1 were upregulated prior to angiogenic sprouting. Exogenous ORM1 delayed the angiogenic response to injury and inhibited the proangiogenic effect of TNFα in cultures of aortic rings or isolated endothelial cells, but stimulated aortic angiogenesis over time while promoting VEGF production and activity. ORM1 inhibited injury- and TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in aortic rings, but not of NFκB. This effect was injury/TNFα-specific since ORM1 did not inhibit VEGF-induced signaling, and cell-specific since ORM1 inhibited TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in macrophages and endothelial cells, but not mural cells. Experiments with specific inhibitors demonstrated that the MEK/ERK pathway was required for angiogenesis. ORM1 inhibited angiogenesis in a subcutaneous in vivo assay of aortic ring-induced angiogenesis, but stimulated developmental angiogenesis in the chorioallantoic membrane (CAM) assay.

Conclusion

ORM1 regulates injury-induced angiogenesis in a time- and context-dependent manner by sequentially dampening the initial TNFα-induced angiogenic response and promoting the downstream stimulation of the angiogenic process by VEGF. The context-dependent nature of ORM1 angioregulatory function is further demonstrated in the CAM assay where ORM1 stimulates developmental angiogenesis without exerting any inhibitory activity.     

Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs

Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.     

Sitagliptin therapy enhances the number of circulating angiogenic cells and angiogenesis—evaluations in vitro and in the rat critical limb ischemia model

Background aimsWe tested the hypothesis that sitagliptin is capable of increasing blood flow in the rat critical limb ischemia (CLI) model by enhancement of angiogenesis.MethodsAdipose tissue from adult-male Fischer 344 rats (n = 6) were cultured in endothelial progenitor cell culture medium for 14 d with (25 μmol/L) or without sitagliptin. CLI was induced by ligation of the left femoral artery. Rats (n = 32) were equally separated into four groups: untreated controls (group 1), sitagliptin (4 mg/kg per day; group 2), CLI (group 3) and CLI with sitagliptin (group 4).ResultsIn vitro, 7 and 14 d after cell culture, endothelial progenitor cell biomarkers assessed by flow cytometry (Sca-1/CD31+, CXCR4+, c-kit+ and CD34+ cells) and Western blot (vascular endothelial growth factor, CXCR4 and stromal-derived factor [SDF]-1α) were remarkably higher in group 4 than in the other groups (all P < 0.01). In vivo, 2 and 14 d after the CLI procedure, circulating angiogenic cell (Sca-1/CD31+, Sca-1+ and CD31+) numbers were significantly higher in group 4 than in the other groups (all P < 0.001). Additionally, the messenger RNA and protein expression of angiogenic biomarkers (CXCR4, SDF-1α and vascular endothelial growth factor), immunofluorescent staining of angiogenic cells (CXCR4+, SDF-1α+, CD31+, von Willebrand factor + cells) and immunohistochemical staining of small vessel numbers in the ischemic area were significantly higher in group 4 than in the other groups (all P < 0.01). Furthermore, laser Doppler showed that the ratio of ischemic/normal blood flow was remarkably higher group 4 than in group 3 by days 14 and 28 after the CLI procedure (all P < 0.01).ConclusionsSitagliptin therapy enhances circulating angiogenic cell numbers, angiogenesis and blood flow in the CLI area.     

The effect of nitrogen containing bisphosphonates,zoledronate and alendronate,on the production of pro-angiogenic factors by osteoblastic cells

Bisphosphonates (BPs) have been shown to influence angiogenesis. This may contribute to BP-associated side-effects such as osteonecrosis of the jaw (ONJ) or atypical femoral fractures (AFF). The effect of BPs on the production of angiogenic factors by osteoblasts is unclear. The aims were to investigate the effect of (1) alendronate on circulating angiogenic factors; vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) in vivo and (2) zoledronate and alendronate on the production of VEGF and ANG-1 by osteoblasts in vitro. We studied 18 post-menopausal women with T score ⩽ −2 randomized to calcium/vitamin D only (control arm, n = 8) or calcium/vitamin D and alendronate 70 mg weekly (treatment arm, n = 10). Circulating concentrations of VEGF and ANG-1 were measured at baseline, 3, 6 and 12 months. Two human osteoblastic cell lines (MG-63 and HCC1) and a murine osteocytic cell line (MLO-Y4) were treated with zoledronate or alendronate at concentrations of 10⁻¹²–10⁻⁶ M. VEGF and ANG-1 were measured in the cell culture supernatant. We observed a trend towards a decline in VEGF and ANG-1 at 6 and 12 months following treatment with alendronate (p = 0.08). Production of VEGF and ANG-1 by the MG-63 and HCC1 cells decreased significantly by 34–39% (p < 0.01) following treatment with zoledronate (10⁻⁹–10⁻⁶ M). Treatment of the MG-63 cells with alendronate (10⁻⁷ and 10⁻⁶) led to a smaller decrease (25–28%) in VEGF (p < 0.05). Zoledronate (10⁻¹⁰–10⁻⁶ M) suppressed the production of ANG-1 by MG-63 cells with a decrease of 43–49% (p < 0.01). Co-treatment with calcitriol (10⁻⁸ M) partially reversed this zoledronate-induced inhibition. BPs suppress osteoblastic production of angiogenic factors. This may explain, in part, the pathogenesis of the BP-associated side-effects.     

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)₄K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)₄K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)₄K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)₄K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Obesity-associated insulin resistance is correlated to adipose tissue vascular endothelial growth factors and metalloproteinase levels

Background

The expansion of adipose tissue is linked to the development of its vasculature, which appears to have the potential to regulate the onset of obesity. However, at present, there are no studies highlighting the relationship between human adipose tissue angiogenesis and obesity-associated insulin resistance (IR).

Results

Our aim was to analyze and compare angiogenic factor expression levels in both subcutaneous (SC) and omentum (OM) adipose tissues from morbidly obese patients (n = 26) with low (OB/L-IR) (healthy obese) and high (OB/H-IR) degrees of IR, and lean controls (n = 17). Another objective was to examine angiogenic factor correlations with obesity and IR. Here we found that VEGF-A was the isoform with higher expression in both OM and SC adipose tissues, and was up-regulated 3-fold, together with MMP9 in OB/L-IR as compared to leans. This up-regulation decreased by 23% in OB/-H-IR compared to OB/L-IR. On the contrary, VEGF-B, VEGF-C and VEGF-D, together with MMP15 was down-regulated in both OB/H-IR and OB/L-IR compared to lean patients. Moreover, MMP9 correlated positively and VEGF-C, VEGF-D and MMP15 correlated negatively with HOMA-IR, in both SC and OM.

Conclusion

We hereby propose that the alteration in MMP15, VEGF-B, VEGF-C and VEGF-D gene expression may be caused by one of the relevant adipose tissue processes related to the development of IR, and the up-regulation of VEGF-A in adipose tissue could have a relationship with the prevention of this pathology.     

Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, K_d, are 0.89 and 0.18 μM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.     

Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis

Using the zebrafish, we previously identified a central function for perlecan during angiogenic blood vessel development. Here, we explored the nature of perlecan function during developmental angiogenesis. A close examination of individual endothelial cell behavior revealed that perlecan is required for proper endothelial cell migration and proliferation. Because these events are largely mediated by VEGF-VEGFR2 signaling, we investigated the relationship between perlecan and the VEGF pathway. We discovered that perlecan knockdown caused an abnormal increase and redistribution of total VEGF-A protein suggesting that perlecan is required for the appropriate localization of VEGF-A. Importantly, we linked perlecan function to the VEGF pathway by efficiently rescuing the perlecan morphant phenotype by microinjecting VEGF-A₁₆₅ protein or mRNA. Combining the strategic localization of perlecan throughout the vascular basement membrane along with its growth factor-binding ability, we hypothesized a major role for perlecan during the establishment of the VEGF gradient which provides the instructive cues to endothelial cells during angiogenesis. In support of this hypothesis we demonstrated that human perlecan bound in a heparan sulfate-dependent fashion to VEGF-A₁₆₅. Moreover, perlecan enhanced VEGF mediated VEGFR2 activation of human endothelial cells. Collectively, our results indicate that perlecan coordinates developmental angiogenesis through modulation of VEGF-VEGFR2 signaling events. The identification of angiogenic factors, such as perlecan, and their role in vertebrate development will not only enhance overall understanding of the molecular basis of angiogenesis, but may also provide new insight into angiogenesis-based therapeutic approaches.     

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

Background

Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis.

Methology/Principal Findings

We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21–66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process.

Conclusions/Significance

Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis.     

Responses of Corpuscles of Stannius to intra-peritoneal vitamin-D3 administration in teleost Labeo rohita (Hamilton, 1822) reared in water with two different levels of calcium concentration

This study assessed the responses of vitamin-D₃ intraperitoneally injected to Rohu, Labeo rohita @ of 0 IU/kg bw (only solvent), 100 IU/kg bw and 500 IU/kg bw reared in 20 and 40 ppm of calcium (Ca) enriched water. The cellular changes in Corpuscles of Stannius (CS) gland, serum Ca, and inorganic phosphate (P_i) level were analysed up to the 60th day. Rohu administered with 100 IU/kg bw D₃ and exposed to 40 ppm Ca-rich water exhibited notable hyperplasia of CS compared with their control groups. Notable changes with high serum Ca level (13.87 ± 0.3 mg/dl) was detected on the 5th day in fish exposed to 40 ppm Ca-rich water, while related values attained (13.74 ± 0.1 mg/dl) only after 7 days in 20 ppm Ca-rich water of 500 IU/kg bw vitamin D₃ injection. Similarly, high serum P_i level (7.66 ± 0.2 mg/dl) in 40 ppm Ca injected with D₃ at 500 IU/kg bw. The results demonstrated that the Ca homeostasis of Labeo rohita is influenced by intra-peritoneal vitamin D₃. Progressive studies should be conducted by increasing the dose of vitamin D₃ to investigate optimum dose/supplement in feed for commercially important aquaculture teleost Labeo rohita for maximum and sustainable absorption of Ca from the variable water Calcium levels to maintain Ca²⁺ homeostasis.     

Chemopreventive effects of vitamin D3 and its analogue,paricalcitol, in combination with 5-fluorouracil against colorectal cancer: The role of calcium signalling molecules

BackgroundAlthough vitamin D (VD) is chemoprotective and enhances 5-fluorouracil (5-FU) cytotoxicity against colorectal cancer (CRC), little is known about its potential calcium (Ca²⁺)-mediated anti-tumorigenic actions. Therefore, this study compared between VD and its non-calcaemic analogue, Paricalcitol (Pcal), ± 5-FU in relation to chemoprevention and Ca²⁺-mediated apoptosis in vivo and in vitro.MethodsSeventy male mice were distributed to: negative controls, positive controls (PC), VD, Pcal, 5-FU, VD + 5-FU and Pcal+5-FU groups. All groups, except negative, received two consecutive azoxymethane (AOM)-injections (10 mg/Kg/week) for CRC induction. VD₃ (1000 IU/kg; three times/week) and Pcal (1.25 μg/kg; three times/week) injections started week-16 post-AOM and for 10 weeks. Three successive 5-FU cycles began at week-21 (50 mg/Kg/week). Similar protocols with VD₃, Pcal and/or 5-FU were applied in the HT29 colon cancer cells.ResultsThe PC group had abundant malignant tumours, markedly elevated proliferation markers (survivin/CCND1) and declines in cyclin-dependent kinase-inhibitor-1A, pro-apoptotic molecules (p53/BAX/cytochrome_C/caspase-3), tissue Ca²⁺ concentrations and Ca²⁺-dependent proteins (CaSR/CAM/CAMKIIA). All monotherapies equally reduced tumour numbers and proliferation markers whilst promoting the anti-tumorigenic molecules. VD and/or 5-FU, but not Pcal monotherapy, enhanced Ca²⁺ levels and Ca²⁺-related molecules (CaSR/CAM/CAMKIIA/BAX/cytochrome_C) in vivo and in vitro. However, VD + 5-FU co-therapy showed the lowest tumour numbers, the highest cell numbers in sub-G1 phase of cell cycle, alongside the most effective modulations of oncogenes, tumour suppressors and Ca²⁺-related molecules at the gene and protein levels in vivo and in vitro.ConclusionsVD₃ was superior than Paricalcitol in potentiating 5-FU cytotoxicity, possibly by upregulating several Ca²⁺-related molecules involved in tumour suppression.     

Vitamin A with L-ascorbic acid sodium salt improves the growth performance,immune function and antioxidant capacity of weaned pigs

Vitamin A is easily degraded by environmental factors. Therefore, it is very important to add antioxidants during Vitamin A production. In the past, ethoxyquin (EQ) was widely used, but recent studies have found that it has potential toxicity. Therefore, in this study, we evaluated the antioxidant activities of 4 antioxidants in vitro: EQ, butylated hydroxytoluene, α-tocopherol and L-ascorbic acid sodium salt (Vitamin C sodium). In vitro experiments showed that Vitamin C sodium had better antioxidant capacity. Then, we explored the effects of different antioxidant types of Vitamin A on the growth performance, immune function and antioxidant capacity of weaned pigs. In total, 288 weaned piglets with an initial mean BW of 8.34 ± 0.02 kg at 30 days old were randomly divided into three groups with four replicates and 24 piglets per replicate for 35 days of feeding. The experimental diets were as follows: i) basal diet without external Vitamin A (NC); ii) basal diet supplemented with 12 000 IU/kg EQ Vitamin A and iii) basal diet supplemented with 12 000 IU/kg Vitamin C sodium Vitamin A. On day 36, two pigs from each replicate were selected to collect serum samples. The in vivo results showed that pigs in the EQ Vitamin A and Vitamin C sodium Vitamin A groups had significantly higher final weight and average daily gain (P < 0.05). During the trial, the levels of IgG and glutathione peroxidase in the EQ Vitamin A and Vitamin C sodium Vitamin A groups were significantly higher than those in the NC group (P < 0.05), and the malondialdehyde content was significantly lower (P < 0.05). On the 36th day, the levels of IgA and total antioxidant capacity in the Vitamin C sodium Vitamin A group were significantly higher than those in the EQ Vitamin A and NC (P < 0.05) groups. Thus, Vitamin C sodium Vitamin A can significantly improve the growth performance, antioxidant capacity and immune function of weaned pigs. Meanwhile, Vitamin C sodium may replace EQ as an antioxidant additive for Vitamin A.     

Molecular cloning of Quek 1 and 2, two quail vascular endothelial growth factor (VEGF) receptor-like molecules

We have previously reported the cloning of two partial cDNAs corresponding to two quail (Coturnix coturnix japonica) receptor tyrosine kinases (RTKs), named Quek 1 and Quek 2, and their expression in endothelial cells of the early avian embryo. We here report the cloning of the full-size cDNAs for both molecules. Sequence comparison shows that Quek 1 and 2 share an overall amino acid (aa) identity of 49%. They both comprise seven extracellular immunoglobulin-like (Ig-like) domains, a single transmembrane domain, and an intracellular kinase domain split into two by a 70 aa insertion. These structural characteristics are shared by the members of the recently discovered VEGF receptor (VEGFR) family. We have compared the sequences of Quek 1 and 2 to the other VEGFRs. At the aa level, Quek 1 is most closely related to KDR/flk-1 (VEGFR 2) (aa identity of 69% and 71%, respectively). Quek 2 shows a similar degree of aa identity to flt-4 (VEGFR 3). Quek 1 and 2 display a lower homology to flt-1 (VEGFR 1) (about 45% aa identity). These data suggest that Quek 1 and 2 are the avian homologues of VEGFRs 2 and 3, respectively.