Attenuation of the exercise‐induced increase in skeletal muscle Flt‐1 mRNA by nitric oxide synthase inhibition

We investigated the vascular endothelial growth factor (VEGF) receptor [fms‐like‐tyrosine kinase (Flt‐1 and fetal liver kinase‐1 (Flk‐1)] response to acute exercise. In female Wistar rats, the VEGF receptor messenger RNA (mRNA) response to a single acute exercise bout was examined using semi‐quantitative Northern blot from the left gastrocnemius muscles at rest and post‐exercise at 0, 1, 2, 4, 8, 16, 24 and 48 h. Exercise altered both Flt‐1 and Flk‐1 mRNA, with significant increases in Flt‐1 mRNA at 1 and 24 h. However, post‐hoc analysis was unable to discern the time point where a significant increase in Flk‐1 mRNA occurred. To investigate the regulation of Flt‐1 mRNA by exercise we examined if nitric oxide synthase (NOS) inhibition alters the Flt‐1 mRNA response. Eight groups [Condition: Rest or Exercise; Drug: Saline, 30 mg kg^–1N^ω‐nitro‐L ‐arginine methyl ester (L ‐NAME), 300 mg kg^–1L ‐NAME or 300 mg kg^–1D ‐NAME] were used to determine the effect of NOS inhibition on the Flt‐1 mRNA response to exercise. L ‐NAME, a known NOS inhibitor, attenuated the exercise‐induced increase in Flt‐1 mRNA by ～50%. These findings suggest that: (1) exercise alters Flt‐1 and Flk‐1 gene expression; and (2) NO is important in the regulation of the Flt‐1 gene response to exercise.     

Sinusoid development and morphogenesis may be stimulated by VEGF‐Flk‐1 signaling during fetal mouse liver development

Early morphogenesis of hepatic sinusoids was histochemically and experimentally analyzed, and the importance of VEGF‐Flk‐1 signaling in the vascular development was examined during murine liver organogenesis. FITC‐gelatin injection experiments into young murine fetuses demonstrated that all primitive sinusoidal structures were confluent with portal and central veins, suggesting that hepatic vessel development may occur via angiogenesis. At 12.5–14.5 days of gestation, VEGF receptors designated Flk‐1, especially their mature form, were highly expressed in endothelial cells of primitive sinusoidal structures and highly phosphorylated on their tyrosine residues. At the same time, VEGF was also detected in hepatoblasts/hepatocytes, hemopoietic cells, and megakaryocytes of the whole liver parenchyma. Furthermore, the addition of VEGF to E12.5 liver cell cultures significantly induced the growth and branching morphogenesis of sinusoidal endothelial cells. Therefore, VEGF‐Flk‐1 signaling may play an important role in the growth and morphogenesis of primitive sinusoids during fetal liver development. Developmental Dynamics 239:386–397, 2010. © 2009 Wiley‐Liss, Inc.     

Alterations in the immunohistochemical distribution patterns of vascular endothelial growth factor receptors Flk1 and Flt1 in bleomycin-induced rat lung fibrosis

To investigate the role of vascular endothelial growth factor (VEGF) in fibrogenesis, the distribution patterns of the VEGF receptors Flt1 and Flk1 were studied by immunohistochemistry, double immunofluorescence, and immunoelectron microscopy in normal (n=2) and bleomycin-treated (n=21) adult rats. Lungs were studied at 5, 24, 28, 35, and 42 days after treatment (p.t.). Flt1, Flk1, and VEGF immunoreactivity localised predominantly to the pulmonary epithelium. In control lungs, Flt1 immunoreactivity was present in ciliated bronchial epithelium and type 2 pneumocytes, Flk1 in Clara cells, and VEGF in Clara cells and type 2 pneumocytes. Flk1 localised to mast cells, present in the peribronchovascular and pleural interstitium only. Flt1- and Flk1-mRNAs were observed in Clara cells and type 2 pneumocytes. Bleomycin-induced fibrogenesis was characterised by a decrease in Flk1 immunoreactivity of Clara cells, and an increase in VEGF-immunoreactive myofibroblasts and type 2 pneumocytes by day 5 p.t., followed by a progressive accumulation of Flk1-immunoreactive mast cells by day 24 p.t. in fibrotic lesions containing VEGF-immunoreactive myofibroblasts. After 42 days, fibrotic regions were densely populated by mast cells. Since mast cells are known to be chemotactically attracted by VEGF, we suggest that VEGF/Flk1 represents the molecular link between proliferation of myofibroblasts, accumulation of mast cells, and the burst of fibrosis at sites of initial lesions in bleomycin-induced fibrosis. Received: 27 August 1998 / Accepted: 31 March 1999     

HIF‐1α inhibition ameliorates an allergic airway disease via VEGF suppression in bronchial epithelium

Hypoxia‐inducible factor‐1α (HIF‐1α) plays a critical role in immune and inflammatory responses. One of the HIF‐1α target genes is vascular endothelial growth factor (VEGF), which is a potent stimulator of inflammation, airway remodeling, and physiologic dysregulation in allergic airway diseases. Using OVA‐treated mice and murine tracheal epithelial cells, the signaling networks involved in HIF‐1α activation and the role of HIF‐1α in the pathogenesis of allergic airway disease were investigated. Transfection of airway epithelial cells with HIF‐1α siRNA suppressed VEGF expression. In addition, the increased levels of HIF‐1α and VEGF in lung tissues after OVA inhalation were substantially decreased by an HIF‐1α inhibitor, 2‐methoxyestradiol. Our data also show that the increased numbers of inflammatory cells, increased airway hyperresponsiveness, levels of IL‐4, IL‐5, IL‐13, and vascular permeability in the lungs after OVA inhalation were significantly reduced by 2‐methoxyestradiol or a VEGF inhibitor, CBO‐P11. Moreover, we found that inhibition of the PI3K p110δ isoform (PI3K‐δ) or HIF‐1α reduced OVA‐induced HIF‐1α activation in airway epithelial cells. These findings indicate that HIF‐1α inhibition may attenuate antigen‐induced airway inflammation and hyperresponsiveness through the modulation of vascular leakage mediated by VEGF, and that PI3K‐δ signaling may be involved in the allergen‐induced HIF‐1α activation.     

Branching of lung epithelium in vitro occurs in the absence of endothelial cells

Background: Early lung morphogenesis is driven by tissue interactions. Signals from the lung mesenchyme drive epithelial morphogenesis, but which individual mesenchymal cell types are influencing early epithelial branching and differentiation remains unclear. It has been shown that endothelial cells are involved in epithelial repair and regeneration in the adult lung, and they may also play a role in driving early lung epithelial branching. These data, in combination with evidence that endothelial cells influence early morphogenetic events in the liver and pancreas, led us to hypothesize that endothelial cells are necessary for early lung epithelial branching. Results: We blocked vascular endothelial growth factor (VEGF) signaling in embryonic day (E) 12.5 lung explants with three different VEGF receptor inhibitors (SU5416, Ki8751, and KRN633) and found that in all cases the epithelium was able to branch despite the loss of endothelial cells. Furthermore, we found that distal lung mesenchyme depleted of endothelial cells retained its ability to induce terminal branching when recombined with isolated distal lung epithelium (LgE). Additionally, isolated E12.5 primary mouse lung endothelial cells, or human lung microvascular endothelial cells (HMVEC‐L), were not able to induce branching when recombined with LgE. Conclusions: Our observations support the conclusion that endothelial cells are not required for early lung branching. Developmental Dynamics 244:553–563, 2015. © 2015 Wiley Periodicals, Inc.     

Regulation of endothelial cell differentiation and arterial specification by VEGF and Notch signaling

Analysis of molecular and cellular mechanisms underlying vascular development in vertebrates indicates that initially vasculogenesis occurs when a primary capillary plexus forms de novo from endothelial cell precursors derived from nascent mesodermal cells. Transplantation experiments in avian embryos demonstrate that embryonic endothelial cells originate from two different mesodermal lineages: splanchnic mesoderm and somites. Genetic analysis of mouse and zebrafish reveals that vascular endothelial growth factor (VEGF)/Flk1 and Notch signaling play crucial roles throughout embryonic vascular development. VEGFA plays a major role in endothelial cell proliferation, migration, survival, and regulation of vascular permeability. Flk1, the primary VEGFA receptor, is the earliest marker of the developing endothelial lineage and is essential for endothelial differentiation during vasculogenesis. Notch signaling has been demonstrated to directly induce arterial endothelial differentiation. Recent studies suggest that Notch signaling is activated downstream of VEGF signaling and negatively regulates VEGF-induced angiogenesis and suppresses aberrant vascular branching morphogenesis. In addition to altering endothelial cell fate through Notch activation, VEGFA directly guides endothelial cell migration in an isoform-dependent manner, modifying vascular patterns. Interestingly, genetic studies in mice show that many molecules involved in VEGF or Notch signaling must be tightly regulated for proper vascular formation. Taken together, VEGF and Notch signaling apparently coordinate vascular patterning by regulating each other.     

Advanced glycation end product Nε‐carboxymethyllysine induces endothelial cell injury: the involvement of SHP‐1‐regulated VEGFR‐2 dephosphorylation

N^ε‐carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes‐induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML‐related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)‐mediated SH2 domain‐containing tyrosine phosphatase‐1 (SHP‐1) activation by CML inhibits the VEGF receptor‐2 (VEGFR‐2, KDR/Flk‐1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR‐2 activation in parallel with the increased SHP‐1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP‐1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP‐1 with VEGFR‐2. Consistently, SHP‐1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [³H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML‐increased SHP‐1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP‐1 and CML was increased, but phospho‐VEGFR‐2 staining was decreased in the aortic endothelium of streptozotocin‐induced and high‐fat diet‐induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP‐1‐regulated VEGFR‐2 dephosphorylation through NADPH oxidase‐derived ROS is involved in the CML‐triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Aquaporin‐1 expression in the chick embryo chorioallantoic membrane

The chick embryo chorioallantoic membrane (CAM) is commonly used in vivo to study both angiogenesis and anti‐angiogenesis. Rapid membrane water transport is mediated by a family of molecular water channels, called aquaporins (AQPs), which have been identified in the epithelial and endothelial cells of higher vertebrates. AQP1, expressed in adsorptive and secretory epithelia, is also expressed in endothelial cells of capillaries and arteries. Its mRNA has been found in vascular smooth muscle cells (VSMCs) of arteries and capillaries, as well as in a subset of VSMCs of human atherosclerotic plaques. This study investigated the developmental expression of AQP1 in the chick CAM by Western blot and immunohistochemistry. Western blot results show that a major nonglycosylated band was observed with electrophoretic mobility of approximately 28 kDa in the three developmental stages examined. Immunohistochemistry data demonstrate that AQP1 was clearly expressed in the ectodermal and endodermal epithelia, the vascular endothelium, and the VSMCs. Because little information is available on the behavior of microvessel AQP1 during angiogenesis in normal and pathological conditions, our data relative to the pattern of expression of AQP1 in CAM blood vessels in normal conditions may be considered a useful tool to further investigate its modifications in several experimental conditions implying a stimulation or an inhibition of angiogenesis in the CAM assay. Anat Rec 268:85–89, 2002. © 2002 Wiley‐Liss, Inc.     

VEGF is deposited in the subepithelial matrix at the leading edge of branching airways and stimulates neovascularization in the murine embryonic lung.

We used whole lung cultures as a model to study blood vessel formation in vitro and to examine the role that epithelial-mesenchymal interactions play during embryonic pulmonary vascular development. Mouse lungs were isolated at embryonic day 11.5 (E11.5) and cultured for up to 4 days prior to blood vessel analysis. Platelet endothelial cell adhesion molecule-1 (PECAM/CD31) and thrombomodulin (TM/CD141) immunolocalization demonstrate that vascular development occurs in lung cultures. The vascular structures identified in lung cultures first appear as a loosely associated plexus of capillary-like structures that with time surround the airways. To investigate the potential role of vascular endothelial cell growth factor (VEGF) during pulmonary neovascularization, we immunolocalized VEGF in embryonic lungs. Our data demonstrate that VEGF is uniformly present in the airway epithelium and the subepithelial matrix of E11.5 lungs. At later time points, E13.5 and E15.5, VEGF is no longer detected in the proximal airways, but is restricted to the branching tips of airways in the distal lung. RT-PCR analysis reveals that VEGF(164) is the predominant isoform expressed in lung cultures. Grafting heparin-bound VEGF(164) beads onto lung explants locally stimulates a marked neovascular response within 48 hr in culture. Semi-quantitative RT-PCR reveals an 18% increase in PECAM mRNA in VEGF(164)-treated whole lung cultures as compared with untreated cultures. The restricted temporal and spatial expression of VEGF suggests that matrix-associated VEGF links airway branching with blood vessel formation by stimulating neovascularization at the leading edge of branching airways.     

PDGF Receptor Alpha+ Mesoderm Contributes to Endothelial and Hematopoietic Cells in Mice

Background: Early mesoderm can be classified into Flk‐1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk‐1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato‐endothelial lineages has not been proved in mouse embryos. Results: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin‐Kit+Sca‐1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk‐1+ population that exists until E8.5 may be the source for hemato‐endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin‐Kit+Sca‐1+ (KSL) cells, respectively. Conclusions: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos. Developmental Dynamics 242:254–268, 2013. © 2013 Wiley Periodicals, Inc.     

A Membrane Associated mCherry Fluorescent Reporter Line for Studying Vascular Remodeling and Cardiac Function During Murine Embryonic Development

The development of the cardiovascular system is a highly dynamic process dependent on multiple signaling pathways regulating proliferation, differentiation, migration, cell–cell and cell‐matrix interactions. To characterize cell and tissue dynamics during the formation of the cardiovascular system in mice, we generated a novel transgenic mouse line, Tg(Flk1::myr‐mCherry), in which endothelial cell membranes are brightly labeled with mCherry, a red fluorescent protein. Tg(Flk1::myr‐mCherry) mice are viable, fertile, and do not exhibit any developmental abnormalities. High levels of mCherry are expressed in the embryonic endothelium and endocardium, and expression is also observed in capillaries in adult animals. Targeting of the fluorescent protein to the cell membrane allows for subcellular imaging and cell tracking. By acquiring confocal time lapses of live embryos cultured on the microscope stage, we demonstrate that the newly generated transgenic model beautifully highlights the sprouting behaviors of endothelial cells during vascular plexus formation. We have also used embryos from this line to imaging the endocardium in the beating embryonic mouse heart, showing that Tg(Flk1::myr‐mCherry) mice are suitable for the characterization of cardio dynamics. Furthermore, when combined with the previously described Tg(Flk1::H2B‐EYFP) line, cell number in addition to cell architecture is revealed, making it possible to determine how individual endothelial cells contribute to the structure of the vessel. Anat Rec, 2008. © 2008 Wiley‐Liss, Inc.     

Tissue‐specific venous expression of the EPH family receptor EphB1 in the skin vasculature

Background : The major arteries and veins are formed early during development. The molecular tools to identify arterial and venous endothelial cells improve our understanding of arterial–venous differentiation and branching morphogenesis. Compared with arterial differentiation, relatively little is known about what controls venous development, due to lack of definitive molecular markers for venous endothelial cells. Results: Here we report that the antibody against EphB1, an EphB class receptor, makes it possible to establish a reliable whole‐mount immunohistochemical analysis of venous identity with greater resolution than previously possible in embryonic and adult skin vasculature models. EphB1 expression is restricted to the entire venous vasculature throughout embryonic development to adulthood, whereas the previously established venous marker EphB4 is also detectable in lymphatic vasculature. This venous‐restricted expression of EphB1 is established after the vascular remodeling of the primary capillary plexus has occurred. Compared with its venous‐specific expression in the skin, however, EphB1 is not restricted to the venous vasculature in yolk sac, trunk and lung. Conclusions: These studies introduce EphB1 as a new venous‐restricted marker in a tissue‐specific and time‐dependent manner. Developmental Dynamics 242:976–988, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival

The formation of new vessels (angiogenesis) is essential for primary tumour growth and metastasis and is induced by several angiogenic factors, including vascular endothelial growth factor (VEGF). The microvascular density (MVD) in tumours was assessed and the expression of VEGF and its receptors VEGF-R1-Flt1 and VEGF-R2-KDR/Flk1 was investigated in the different cellular compartments in vivo, in order to establish their interrelationship and their prognostic influence. Immunohistochemical study of 69 stage I–II non-small cell lung carcinomas (NSCLCs) was performed on paraffin sections with CD34 antibody to estimate MVD, using a Chalkley eye-piece graticule and VEGF, VEGF-R1, and VEGF-R2 antibodies. There was strong expression of VEGF and its receptors in tumour cells, endothelial cells, and stromal fibroblasts. In tumour cells, the level of VEGF was correlated with that of VEGF-R1 ( p = 0·018) but not that of VEGF-R2. In fibroblasts, high expression of VEGF was correlated with that of VEGF-R1 ( p = 0·0001) and VEGF-R2 ( p = 0·0001). In endothelial cells, expression of VEGF was correlated with that of VEGF-R1 ( p < 0·0001) and VEGF-R2 ( p = 0·04). The level of VEGF in fibroblasts was correlated with that of VEGF-R1 ( p = 0·0028) and VEGF-R2 ( p = 0·01) in endothelial cells. There was no correlation between the level of MVD and that of VEGF or VEGF-R1 or VEGF-R2. Neither the level of MVD, nor the level of expression of VEGF and VEGF receptors in any compartment influenced the patient's survival. In conclusion, although angiogenesis is essential for tumour growth, this study failed to demonstrate that MVD, VEGF, VEGF-R1, and VEGF-R2 are prognostic markers for stage I–II NSCLC. VEGF, however, might act as a direct autocrine growth factor for tumour cells via VEGF-R1 and angiogenesis could be promoted in a paracrine loop, where VEGF is produced by fibroblasts and tumour cells and then binds to endothelial cells via induced VEGF receptors. VEGF and its receptors thus appear as relevant therapeutic targets in NSCLC. Copyright © 1999 John Wiley & Sons, Ltd.     

Role of Etv2‐positive cells in the remodeling morphogenesis during vascular development

Etv2 is a critical determinant for the commitment of endothelial (EC) and hematopoietic (HPC) cells from mesoderm. Etv2 is assumed to be transiently required for EC commitment but dispensable after most ECs differentiate around E9.5. To confirm the time window of Etv2 requirement, Etv2 was ablated at different time points using ROSA26CreER mice. Unexpectedly, Etv2 ablation at E9.5 caused vascular remodeling defects in cranial and yolk sac vasculature. Immunostaining showed that Etv2+/VE‐cadherin (VECAD)− cells were present around forming vasculature, mostly co‐expressing Flk‐1 with a small number of Etv2+/VECAD+ cells, indicating that Etv2+/Flk‐1+/VECAD− cells are the major Etv2+ population promoting vascular remodeling around E9.5. Gene expression analysis showed up‐regulation of Fgf proteins, Il‐6, Glypican‐3 and matrix metalloproteases in Etv2+/VEDAC− cells over Etv2−/VECAD+ mature ECs. Blockade of those factors caused reduced EC sprouting in ex vivo explant culture from E9.5 embryos, suggesting the functional significance of environmental factors derived from Etv2+ cells. Altogether, we propose that Etv2+/VEDAC− cells around E9.5‐E10.5 provide extracellular factors to complete vascular morphogenesis in addition to becoming differentiated ECs incorporated into vessels. This insight for the new role of Ets protein in perivascular Flk‐1+/VECAD−/(Etv2+) cells to induce expression of angiogenic factors may provide another strategy to control angiogenesis.     

Cartilage canals in the chicken embryo are involved in the process of endochondral bone formation within the epiphyseal growth plate

A detailed study of so-called communicating cartilage canals, which penetrate deeply up into the lower hypertrophic zone of the epiphyseal growth plate in the embryonic chicken femur (E20), was carried out with the aim to clarify whether or not these canals are involved in the bone-forming process. In addition, we examined the manner in which cartilage canals are formed and compare the present data with our previous data. The canals were investigated by means of light microscopy, electron microscopy, immunohistochemistry (VEGF, VEGFR2/Flk1, type I collagen), and 3D reconstruction. Some communicating canals deeply penetrate into the upper hypertrophic zone where they terminate, showing electron-dense cells at their end. Subcellular characteristics of these cells are hardly detectable and we suppose that they undergo cell death. Other canals pass down deeper into the lower hypertrophic zone. The upper segment of these canals is composed of capillaries, mesenchymal cells, and macrophage-like cells. Precursors of osteoblasts are adjacent to the canals. The lower segment of communicating canals is composed of bone matrix or osteoid, which contains type I collagen fibrils and cells having the typical subcellular features of osteoblasts. No vessels are found in these segments. Immunohistochemistry shows that the matrix of the canals labels positively for type I collagen. In addition, staining with sirius red demonstrates that bone matrix is formed in these parts. We assume that the osteoblast-like cells of the lower segments of communicating canals originate either from mesenchymal cells or even from hypertrophic chondrocytes. Our immunohistochemical data also reveal that vascular endothelial growth factor (VEGF) and the corresponding receptor VEGFR2/Flk1 (VEGF receptor 2/Flk1) are localized in cartilage canals of the reserve zone, the proliferative zone, and the hypertrophic zone. The receptor is found in the endothelial cells of the vessels. Furthermore, VEGF is present in hypertrophic chondrocytes. The results of our study suggest that cartilage canals penetrate actively into the cartilage anlage and that bone is formed in the lower segments of the communicating canals where no vessels are detectable.     

Engineering surfaces for site-specific vascular differentiation of mouse embryonic stem cells

Differentiation of stem and progenitor cells routinely relies on the application of soluble growth factors, an approach that enables temporal control of cell fate but enables no spatial control of the differentiation process. Angiogenic progenitor cells derived from mouse embryonic stem cells (ESCs) were differentiated here according to the pattern of immobilized vascular endothelial growth factor-A (VEGF). Mouse ESCs engineered to express green fluorescent protein (eGFP) under control of promoter for the receptor tyrosine kinase Flk1 were used. The Flk1+ angiogenic progenitors were selected from day 3 differentiating embryoid bodies based on their expression of eGFP using fluorescence activated cell sorting. Mouse VEGF₁₆₅ was covalently immobilized onto collagen IV (ColIV) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. A non-cell adhesive layer of photocrosslinkable chitosan was first created, after which VEGF–ColIV was stamped as 100 μm wide lanes on top of the chitosan layer and the Flk1+ angiogenic progenitors were seeded for site-specific differentiation. Lanes stamped with only ColIV served as controls. The results presented here demonstrate that the cultivation of Flk1+ progenitors on surfaces with immobilized VEGF yielded primarily endothelial cells (53 ± 13% CD31 positive and 17 ± 2% smooth muscle actin positive), whereas surfaces without VEGF favored vascular smooth muscle-like cell differentiation (26 ± 17% CD31 positive and 38 ± 9% smooth muscle actin positive).     

Impaired Cytoplasmic–Nuclear Transport of Hypoxia‐Inducible Factor‐1α in Amyotrophic Lateral Sclerosis

We investigated the mechanisms underlying abnormal vascular endothelial growth factor (VEGF) production in amyotrophic lateral sclerosis (ALS). We immunohistochemically studied VEGF, its receptors VEGFR1 and 2, and hypoxia‐inducible factor‐1α (HIF‐1α) in autopsied ALS spinal cords. We also chronologically assessed the expression of HIF‐1α, karyopherin β1, karyopherin β‐cargo protein complex inhibitors and nuclear pore complex proteins in G93A mutant superoxide dismutase 1 (mSOD1) transgenic mice at presymptomatic, symptomatic and end stages. In ALS patients, compared with controls, HIF‐1α immunoreactivity in the cytoplasm of anterior horn cells (AHCs) was significantly increased, while immunoreactivities for VEGF and VEGFRs were significantly decreased. Similar changes in HIF‐1α and VEGF levels were observed in mSOD1 transgenic mice. HIF‐1α co‐localized with karyopherin β1 in the cytoplasm of AHCs and karyopherin β1 co‐localized with nucleoporin 62 (Nup62) on the nuclear envelope. From the presymptomatic stage of mSOD1 transgenic mice, karyopherin β1 immunoreactivity in AHC nuclei significantly decreased and morphological irregularities of the Nup62‐immunostained nuclear envelope became more pronounced with disease progression. Thus, in AHCs from mSOD1 transgenic mice, transport of cytoplasmic HIF‐1α to the nuclear envelope and into the nucleus is impaired from the presymptomatic stage, suggesting that impaired cytoplasmic–nuclear transport of HIF‐1α through the nuclear pore might precede motor neuron degeneration.     

The role of high‐mobility group box protein 1 in collagen antibody‐induced arthritis is dependent on vascular endothelial growth factor

High‐mobility group box 1 (HMGB1) has been implicated in angiogenesis and rheumatoid arthritis (RA). The aim of this study was to define more clearly the role of HMGB1 in the synovial angiogenesis and pathogenesis of an immune model of arthritis. BALB/c mice were injected with monoclonal anti‐collagen antibody cocktail followed by lipopolysaccharide to induce arthritis. HMGB1 and vascular endothelial growth factor (VEGF) were over‐expressed in the areas of the synovium where more inflammation and neoangiogenesis were present. The selective blockade of HMGB1 or VEGF resulted alternatively in a lower severity of arthritis evaluated by the arthritis index. Furthermore, exogenous HMGB1 administration caused a worsening of arthritis, associated with VEGF up‐regulation and increased synovial angiogenesis. The selective inhibition of VEGF also resulted in no induction of arthritis in mice receiving exogenous HMGB1. Cytokine enzyme‐linked immunosorbent assay (ELISA) analyses performed on peripheral blood and synovial fluid demonstrated a significant reduction of interleukin (IL)?1β, IL‐6 and tumour necrosis factor (TNF)‐α in mice where HMGB1 and VEGF pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL‐17A concentration. The development of arthritis mediated by HMGB1 and the synovial angiogenesis can be blocked by inhibiting the VEGF activity. The proinflammatory and proangiogenic cytokine IL‐17A was increased when HMGB1 is inhibited, but the synovial angiogenesis was nevertheless reduced in this model of arthritis. Taken together, these findings shed new light on the role of this nuclear protein in the pathogenesis of arthritis in an RA‐like model.