Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

C-reactive protein decreases expression of VEGF receptors and neuropilins and inhibits VEGF165-induced cell proliferation in human endothelial cells

C-reactive protein (CRP) is associated with cardiovascular disease. However, its biological functions for the vascular system are largely unknown. The objective of this study was to determine whether CRP could affect endothelial cell proliferation and expression of VEGF receptors (VEGFRs) and/or neuropilins. Human coronary artery endothelial cells (HCAECs) treated with CRP showed a significant reduction of mRNA levels of VEGFR-2, VEGFR-3, NRP-1, and NRP-2 by 34%, 63%, 41%, and 43%, respectively, as compared to untreated control cells (p < 0.05) by real-time PCR analysis. In addition, VEGF165-induced cell proliferation was determined by [3H]thymidine incorporation and MTS assay as well as capillary-like tube formation on Matrigel. HCAECs pretreated with CRP significantly decreased VEGF165-induced [3H]thymidine incorporation by 73%, MTS absorbance by 44%, and capillary-like tube formation by 54% as compared to CRP-untreated cells (p < 0.05). These data demonstrate that CRP significantly attenuates VEGF165-induced HCAEC proliferation and capillary-like tube formation through downregulation of expression of VEGFRs and NRPs. This study suggests a new molecular mechanism underlying the adverse effect of CRP on the vascular system.     

Interaction of the Coxsackie and adenovirus receptor (CAR) with the cytoskeleton: binding to actin

The Coxsackie and adenovirus receptor (CAR) is a cell adhesion molecule that is highly expressed in the developing brain. CAR is enriched in growth cone particles (GCP) after subcellular fractionation. In GCP, we identified actin as an interaction partner of the cytoplasmic domain of CAR. In vivo, actin and CAR co-immunoprecipitate and co-localize. In vitro, the binding is direct, with a K(d) of approximately 2.6 microM, and leads to actin bundling. We previously demonstrated that CAR interacts with microtubules. These data suggest a role for CAR in processes requiring dynamic reorganization of the cytoskeleton such as neurite outgrowth and cell migration.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis

The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.     

Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis

Using suppression subtractive hybridisation (SSH), we identified a hitherto unreported gene PHACTR-1 (Phosphatase Actin Regulating Protein-1) in Human Umbilical Vascular Endothelial Cells (HUVECs). PHACTR-1 is an actin and protein phosphatase 1 (PP1) binding protein which is reported to be highly expressed in brain and which controls PP1 activity and F-actin remodelling. We have also reported that its expression is dependent of Vascular Endothelial Growth Factor (VEGF-A₁₆₅). To study its function in endothelial cells, we used a siRNA strategy against PHACTR-1. PHACTR-1 siRNA-treated HUVECs showed a major impairment of tube formation and stabilisation. PHACTR-1 depletion triggered apoptosis through death receptors DR4, DR5 and FAS, which was reversed using death receptor siRNAs or with death receptor-dependent caspase-8 siRNA. Our findings suggest that PHACTR-1 is likely to be a key regulator of endothelial cell function properties. Because of its central role in the control of tube formation and endothelial cell survival, PHACTR-1 may represent a new target for the development of anti-angiogenic therapy.     

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.     

Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice

Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2^−/− than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2^−/− mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2^−/− mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2^−/− EPC intramyocardially into mice with induced MI. Per2^−/− reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2^−/− EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.     

The transcription factor Grainyhead like 3 (GRHL3) affects endothelial cell apoptosis and migration in a NO-dependent manner

Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.     

Platelet endothelial cell adhesion molecule‐1 (PECAM1) plays a critical role in the maintenance of human vascular endothelial barrier function

Cardiovascular endothelial barrier dysfunction is associated with a number of cardiovascular diseases. This study aims to investigate the role of platelet endothelial cell adhesion molecule‐1 (PECAM1) in the maintenance of the vascular endothelial barrier integrate. Human umbilical vein endothelial cells (HUVECs) were cultured into monolayers using as an in vitro model to assess the endothelial barrier function. Knockdown of the gene of PECAM1 markedly reduced the transendothelial resistance and increased the permeability of the HUVEC monolayers. From the wild HUVECs, we detected a complex of PECAM1, claudin1, occluding and endothelial cell selective adhesion molecule (ESAM); such a complex was not detected in the PECAM1‐deficient HUVECs. Knockdown of either claudin1, or occludin, or ESAM, did not affect the formation of the tight junction (TJ) complex. Exposure to recombinant interleukin (IL)‐13 inhibited the expression of PECAM1 and down‐regulated the HUVEC monolayer barrier function. PECAM1 plays an important role in the formation of TJ complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Erbin plays a critical role in human umbilical vein endothelial cell migration and tubular structure formation via the Smad1/5 pathway

Angiogenesis is an important process in atherosclerosis. ErbB2 was proved to have an important role in vascular development, but it is still unclear whether Erbin expresses in vessels as well as its location and function in the vessels. In the current study, we investigated the location and function of Erbin in human umbilical veins. The human umbilical veins were prepared, and immunofluorescent analysis was performed to determine the expression of Erbin. Human umbilical vein endothelial cells (HUVECs) were cultured and the lentivirus (LV) containing Erbin RNAi was also prepared. After transfection with the lentivirus, CCK-8 assay and Annexin V-PI assay were used for cell proliferation and apoptosis, respectively. Cell migration was studied using the scratch wound healing assay and the transwell assay. The capillary-like tube formation assay was performed to illustrate the effect of Erbin on HUVEC tube formation. Expression of signaling pathway molecules was assessed with Western blot. The immunofluorescent analysis suggested that Erbin expressed in human umbilical veins and the majority of the Erbin is strongly colocalized in endothelial cells. Although knockdown of Erbin did not affect HUVEC proliferation and apoptosis, it significantly suppressed HUVEC migration and tubular structure formation. Erbin knockdown showed no effect on the ERK1/2 and Smad2/3 signaling pathways but significantly promoted Smad1/5 phosphorylation and nuclear translocation. Ablation of the Smad1/5 pathway decreased the effects of Erbin on endothelial cells. Erbin is mainly localized in endothelial cells in human umbilical veins and plays a critical role in endothelial cell migration and tubular formation via the Smad1/5 pathway.     

Coxsackievirus and adenovirus receptor (CAR) is expressed in male germ cells and forms a complex with the differentiation factor JAM-C in mouse testis

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.     

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell-cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood-testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFα treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli-germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.     

腺苷对人脐静脉内皮细胞三维管状结构形成的影响

目的:探讨腺苷对脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)三维管状结构形成的影响.方法:建立上、下两层内皮细胞,中间为胶原凝胶的三维培养方式.设对照和试验各3孔.对照孔不加腺苷,实验孔内加入10-4mol/L腺苷.观察并记录特定视野下芽生的管状结构数目.结果:HUVEC可以在Ⅰ型胶原凝胶中形成三维网状结构,腺苷实验组细胞生长快,出芽快,管状结构粗大,甚可形成贯穿胶原的三维网状结构.血管芽生数与对照组比较在24 h、48 h、72 h、96 h均有统计学差异(P＜0.05或P＜0.01).结论:腺苷对HUVEC三维网状结构的形成有促进作用.     

Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28 , 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time a functional interaction between NCAM and EGFR in mammalian cells and investigates the molecular mechanisms underlying this interaction. First, NCAM and EGFR are shown to play opposite roles in neurite outgrowth regulation in cerebellar granular neurons. The data presented indicate that negative regulation of EGFR is one of the mechanisms underlying the neuritogenic effect of NCAM. Second, it is demonstrated that expression of the NCAM-180 isoform induces EGFR down-regulation in transfected cells and promotes EGFR down-regulation induced by EGF stimulation. It is demonstrated that the mechanism underlying this NCAM-180-induced EGFR down-regulation involves increased EGFR ubiquitination and lysosomal EGFR degradation. Furthermore, NCAM-180-mediated EGFR down-regulation requires NCAM homophilic binding and interactions of the cytoplasmic domain of NCAM-180 with intracellular interaction partners, but does not require NCAM-mediated fibroblast growth factor receptor activation.