A new, rapid and reproducible method to obtain high quality endothelium in vitro

Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

人脐静脉内皮细胞的原代培养和鉴定

目的：探索建立稳定有效的脐静脉内皮细胞体外培养方法。方法：用0．1％II型胶原酶消化分离人脐静脉肉皮细胞,加入含内皮细胞生长因子的M199完全培养液中培养,用胰蛋白酶-EDTA进行消化传代培养,用光镜和免疫组化方法对培养的细胞进行形态观察和鉴定。结果：原代培养的内皮细胞在接种后24h后完全贴壁生长,第445天后融合呈铺路石样镶嵌排列,免疫组化可见胞浆中第Ⅷ因子相关抗原呈阳性反应,证实培养的细胞为内皮细胞。结论：脐静脉灌注II型胶原酶消化法配合M199完全培养液可获得高纯度的内皮细胞,细胞可传代5—6次,但细胞产出量不高,不能传10代以上,5代以后细胞形态变化较大,对于复杂的基础研究应用受限。     

Primary cilia of human endothelial cells disassemble under laminar shear stress

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-alpha-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.     

The small GTPase Cdc42 is necessary for primary ciliogenesis in renal tubular epithelial cells

Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis.     

CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease

Primary cilia are nonmotile organelles implicated in signaling and sensory functions. Understanding how primary cilia assemble could shed light on the many human diseases caused by mutations in ciliary proteins. The centrosomal protein CP110 is known to suppress ciliogenesis through an unknown mechanism. Here, we report that CP110 interacts with CEP290--a protein whose deficiency is implicated in human ciliary disease--in a discrete complex separable from other CP110 complexes involved in regulating the centrosome cycle. Ablation of CEP290 prevents ciliogenesis without affecting centrosome function or cell-cycle progression. Interaction with CEP290 is absolutely required for the ability of CP110 to suppress primary cilia formation. Furthermore, CEP290 and CP110 interact with Rab8a, a small GTPase required for cilia assembly. Depletion of CEP290 interferes with localization of Rab8a to centrosomes and cilia. Our results suggest that CEP290 cooperates with Rab8a to promote ciliogenesis and that this function is antagonized by CP110.     

A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo

Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPase-regulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.     

The PCP effector Fuzzy controls cilial assembly and signaling by recruiting Rab8 and Dishevelled to the primary cilium

The planar cell polarity (PCP) pathway controls multiple cellular processes during vertebrate development. Recently the PCP pathway was implicated in ciliogenesis and in ciliary function. The primary cilium is an apically projecting solitary organelle that is generated via polarized intracellular trafficking. Because it acts as a signaling nexus, defects in ciliogenesis or cilial function cause multiple congenital anomalies in vertebrates. Loss of the PCP effector Fuzzy affects PCP signaling and formation of primary cilia; however, the mechanisms underlying these processes are largely unknown. Here we report that Fuzzy localizes to the basal body and ciliary axoneme and is essential for ciliogenesis by delivering Rab8 to the basal body and primary cilium. Fuzzy appears to control subcellular localization of the core PCP protein Dishevelled, recruiting it to Rab8-positive vesicles and to the basal body and cilium. We show that loss of Fuzzy results in inhibition of PCP signaling and hyperactivation of the canonical WNT pathway. We propose a mechanism by which Fuzzy participates in ciliogenesis and affects both canonical WNT and PCP signaling.     

Centrin2 regulates CP110 removal in primary cilium formation

Primary cilia are antenna-like sensory microtubule structures that extend from basal bodies, plasma membrane–docked mother centrioles. Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood. We used reverse genetics to test the role of the small calcium-binding protein, centrin2, in ciliogenesis. Primary cilia arise in most cell types but have not been described in lymphocytes. We show here that serum starvation of transformed, cultured B and T cells caused primary ciliogenesis. Efficient ciliogenesis in chicken DT40 B lymphocytes required centrin2. We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110. Knockdown of CP110 rescued ciliation in CETN2-deficient cells. Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.     

Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish

In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.     

Syndecan-4 Is a Major Syndecan in Primary Human Endothelial Cells In Vitro,Modulated by Inflammatory Stimuli and Involved in Wound Healing

Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1β (IL-1β). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.     

Endothelial primary cilia inhibit atherosclerosis

Primary cilia are microtubule‐based structures present on most mammalian cells that are important for intercellular signaling. Cilia are present on a subset of endothelial cells where they project into the vessel lumen and are implicated as mechanical sensors of blood flow. To test the in vivo role of endothelial cilia, we conditionally deleted Ift88, a gene required for ciliogenesis, in endothelial cells of mice. We found that endothelial primary cilia were dispensable for mammalian vascular development. Cilia were not uniformly distributed in the mouse aorta, but were enriched at vascular branch points and sites of high curvature. These same sites are predisposed to the development of atherosclerotic plaques, prompting us to investigate whether cilia participate in atherosclerosis. Removing endothelial cilia increased atherosclerosis in Apoe^?/? mice fed a high‐fat, high‐cholesterol diet, indicating that cilia protect against atherosclerosis. Removing endothelial cilia increased inflammatory gene expression and decreased eNOS activity, indicating that endothelial cilia inhibit pro‐atherosclerotic signaling in the aorta.     

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.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

CCRK depletion inhibits glioblastoma cell proliferation in a cilium‐dependent manner

Loss of primary cilia is frequently observed in tumour cells, including glioblastoma cells, and proposed to benefit tumour growth, but a causal link has not been established. Here, we show that CCRK (cell cycle‐related kinase) and its substrate ICK (intestinal cell kinase) inhibit ciliogenesis. Depletion of CCRK leads to accumulation of ICK at ciliary tips, altered ciliary transport and inhibition of cell cycle re‐entry in NIH3T3 fibroblasts. In glioblastoma cells with deregulated high levels of CCRK, its depletion restores cilia through ICK and an ICK‐related kinase MAK, thereby inhibiting glioblastoma cell proliferation. These results indicate that inhibition of ciliogenesis might be a mechanism used by cancer cells to provide a growth advantage.     

Rabs and other small GTPases in ciliary transport

The non-motile primary cilium is a single, microtubule-based hair-like projection that emanates from most, if not all, non-dividing mammalian cells. Enriched in a variety of signalling receptors and accessories, the cilium mediates crucial sensory and regulatory functions during development and postnatal tissue homoeostasis. Maintenance of ciliary morphology and function requires continuous IFT (intraflagellar transport), and recent findings have shed light on some molecular details of how ciliogenesis is dependent on targeted exocytic membrane trafficking from the Golgi. The ARL [Arf (ADP ribosylation factor)-related] small GTPase Arf4 functions in TGN (trans-Golgi network) sorting of cilia-targeted rhodopsin into carrier vesicles, while Arl6 (Arf-like 6) and Arl13b regulate aspects of ciliary transport and IFT. Ciliogenesis and ciliary functions are also regulated by small Rabs. Rab8a, in conjunction with Rab11a, and via its interaction with a multitude of proteins associated with the ciliary basal body and axoneme/membrane, appears to be critical for ciliogenesis. Rab8's close homologue Rab10 may also play a ciliogenic role in some cells. Rab23, the depletion or inactivation of which affects cilia formation, may regulate specific ciliary protein targeting and turnover, particularly those involved in Shh (Sonic hedgehog) signalling. Recent findings have also implicated Ran, a small GTPase better known for nuclear import, in ciliary targeting of the KIF17 motor protein. We highlight and discuss recent findings on how Rabs and other small GTPases mediate ciliogenesis and ciliary traffic.     

ICK is essential for cell type‐specific ciliogenesis and the regulation of ciliary transport

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK‐deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK‐deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT‐A, IFT‐B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT‐B, but not IFT‐A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.     

In vivo ciliogenesis in human fetal tracheal epithelium

Development of ciliated cells (CC) in the fetal human trachea was studied by light and electron microscopy in specimens obtained from 45 embryos or fetuses aged from 9 to 27 weeks of gestation (menstrual age). Four stages could be recognized during tracheal development. Up to 11 weeks (stage I), the trachea was covered with a columnar undifferentiated epithelium with abundant glycogen, apical microvilli, and primary cilia. From 12 to 18-19 weeks (stage II), centriologenesis and secondary ciliogenesis were very active, and the percentage of CC and secretory cells (SC) progressively increased. From 20 to 22-23 weeks, the density of CC was higher but, in parallel, the percentage of SC decreased (stage III). Throughout this period, the different steps of ciliogenesis could be identified in the same field, and the ciliated borders consisted of ciliary shafts with a disorderly arrangement. Megacilia were identified. Some of the preciliated cells had both cilia and secretory granules in their apical cytoplasm. After 24 weeks (stage IV), the ciliated border was apparently mature, the rootlets lengthened, and the cilia were correctly orientated. Whatever the fetal age, the density of CC was significantly higher (P less than .01) in the dorsal trachea compared to the ventral trachea. There are many similarities between animal and human ciliogenesis, but in human fetuses, most of the ciliary differentiation occurs early, during the first half of gestation. As demonstrated in experimental models, SC likely play a major role in genesis of CC during the fetal development of the human trachea.     

A role for the primary cilium in Notch signaling and epidermal differentiation during skin development

Ciliogenesis precedes lineage-determining signaling in skin development. To understand why, we performed shRNA-mediated knockdown of seven intraflagellar transport proteins (IFTs) and conditional ablation of Ift-88 and Kif3a during embryogenesis. In both cultured keratinocytes and embryonic epidermis, all of these eliminated cilia, and many (not Kif3a) caused hyperproliferation. Surprisingly and independent of proliferation, ciliary mutants displayed defects in Notch signaling and commitment of?progenitors to differentiate. Notch receptors and Notch-processing enzymes colocalized with cilia in wild-type epidermal cells. Moreover, differentiation defects in ciliary mutants were cell autonomous and rescued by activated Notch (NICD). By contrast, Shh signaling was neither operative nor required for?epidermal ciliogenesis, Notch signaling, or differentiation. Rather, Shh signaling defects in ciliary mutants occurred later, arresting hair follicle morphogenesis in the skin. These findings unveil temporally and spatially distinct functions for primary cilia?at the nexus of signaling, proliferation, and differentiation.