Tube morphogenesis: making and shaping biological tubes

Many organs are composed of epithelial tubes that transport vital fluids. Such tubular organs develop in many different ways and generate tubes of widely varying sizes and structures, but always with the apical epithelial surface lining the lumen. We describe recent progress in several diverse cell culture and genetic models of tube morphogenesis, which suggest apical membrane biogenesis, vesicle fusion, and secretion play central roles in tube formation and growth. We propose a unifying mechanism of tube morphogenesis that has been modified to create tube diversity and describe how defects in the tube size-sensing step can lead to polycystic kidney disease.     

How to shape a cylinder: pollen tube as a model system for the generation of complex cellular geometry

Expansive growth in plant cells is a formidable problem for biophysical studies, and the mechanical principles governing the generation of complex cellular geometries are still poorly understood. Pollen, the male gametophyte stage of the flowering plants, is an excellent model system for the investigation of the mechanics of complex growth processes. The initiation of pollen tube growth requires first of all, the spatially confined formation of a protuberance. This process must be controlled by the mechanical properties of the cell wall, since turgor is a non-vectorial force. In the elongating tube, cell wall expansion is confined to the apex of the cell, requiring the tubular region to be stabilized against turgor-induced tensile stress. Tip focused surface expansion must be coordinated with the supply of cell wall material to this region requiring the precise, logistical control of intracellular transport processes. The advantage of such a demanding mechanism is the high efficiency it confers on the pollen tube in leading an invasive way of life.     

Effects of glucose on migration, proliferation and tube formation by vascular endothelial cells.

In order to elucidate the association between hyperglycemia and the vascular complications of diabetes, the effects of high glucose concentrations on the migration, proliferation and tube formation of bovine carotid artery endothelial cells were investigated. Cells treated with 16.7 and 33.3 mM glucose for 6 days showed 1.69- and 1.75-fold increase in serum-induced migration compared with cells treated with 5.6 mM glucose (p less than 0.05). The effect of glucose on cell proliferation was affected by serum concentration. When this was below 0.5%, a high glucose concentration stimulated cell growth to a maximum of 1.73 times that at a serum concentration of 0.05% (p less than 0.01) whereas at a serum concentration of 10%, growth was inhibited (p less than 0.05). Tube formation was studied by culturing the cells between two layers of collagen gel. Ultrastructurally, tubular structures were composed of one to several endothelial cells containing pinocytotic vesicles and cytoplasmic projections, and linked by junctional complexes. A basal lamina-like structure surrounded the abluminal surface. Treatment of the cells with 16.7 and 27.8 mM glucose for 4 days stimulated tubular elongation 1.85 and 1.71 times, respectively (p less than 0.01). Other osmogenic molecules such as mannitol and sucrose did not affect tube formation. These data imply that high glucose concentrations mimicking diabetic hyperglycemia may not inhibit the repair of endothelial injury and could act as a stimulator of neovascularization.     

十五肽BPC-157对人脐静脉内皮细胞功能的影响

目的:观察十五肽BPC-157对人脐静脉内皮细胞株HUVEC增殖、周期、迁移及小管形成的影响。方法:用不同浓度(0、1、5、10、50、100μg/mL)的BPC-157作用于HUVEC细胞株,采用MTT法检测药物对内皮细胞增殖的影响,通过流式细胞仪观察细胞周期的变化,经细胞划痕和Transwell实验检测药物对内皮细胞迁移的影响,并且通过小管形成实验观察BPC-157对内皮细胞小管形成能力的影响。结果:HUVEC细胞株经BPC-157刺激48 h后,细胞增殖率和各时期细胞比例没有明显变化;而在刺激12 h时,BPC-157显著性促进细胞伤口愈合及穿膜细胞数的增加(P0.01);刺激8 h时,给药组细胞开始聚合,形成复杂的管状网络结构,特别是5μg/mL剂量组。结论:十五肽BPC-157对人脐静脉内皮细胞株HUVEC增殖及细胞周期的改变基本没有影响,但对内皮细胞的迁移及小管形成能力具有明显的促进作用。     

From fate to function: the Drosophila trachea and salivary gland as models for tubulogenesis

Tube formation is a ubiquitous process required to sustain life in multicellular organisms. The tubular organs of adult mammals include the lungs, vasculature, digestive and excretory systems, as well as secretory organs such as the pancreas, salivary, prostate, and mammary glands. Other tissues, including the embryonic heart and neural tube, have requisite stages of tubular organization early in development. To learn the molecular and cellular basis of how epithelial cells are organized into tubular organs of various shapes and sizes, investigators have focused on the Drosophila trachea and salivary gland as model genetic systems for branched and unbranched tubes, respectively. Both organs begin as polarized epithelial placodes, which through coordinated cell shape changes, cell rearrangement, and cell migration form elongated tubes. Here, we discuss what has been discovered regarding the details of cell fate specification and tube formation in the two organs; these discoveries reveal significant conservation in the cellular and molecular events of tubulogenesis.     

Inhibitory effects of novel integrin-targeting peptides on angiogenesis activity in HUVEC cells in vitro

Integrins are critically involved in many tumour-promoting activities. The development of inhibitors against integrins may suppress tumour growth by inhibiting tumour angiogenic signalling. In this study, we investigated the effects of two novel peptides containing the integrin binding arginine-glycine-aspartic acid-motif on inhibiting diverse cell behaviours, including cell adhesion, motility, invasion, tube formation and cell cytoskeleton. Cell adhesion and motility assays demonstrated that cyclopeptides c-Gly and c-Lys might inhibit the adhesive and motile activity at the concentration of 25 μM. There was no significant effect on cell invasion, indicating the importance of extracellular matrix degradation in modulating the anti-invasive effect of human umbilical vein endothelial cells (HUVECs). More importantly, the tubular network formation of HUVECs was significantly inhibited by cyclopeptide c-Lys besides causing a remarkable inhibition of cytoskeletal organization, disrupting the focal adhesion and actin stress fibres formation. In conclusion, this study results indicated that the novel peptide c-Lys has the ability to inhibit diverse cell behaviours of HUVECs, and the effects may be mediated at different levels of the tumour growth. Therefore, c-Lys is perhaps proposed to be a potent anti-angiogenic drug candidate.     

Effect of the extracellular matrix molecules fibronectin and laminin on the adhesion and growth of primary renal cortical epithelial cells

Primary tubular epithelial cells were isolated from renal cortex following enzymatic dissociation with collagenase. These cells were then grown in chemically defined media containing insulin, transferrin, selenium, tri-iodothyronine and either fibronectin or laminin. The tubular epithelial cells were studied ultrastructurally and compared to another epithelial cell type present in the renal cortex, the glomerular epithelial cell. In contrast to the constant morphology of glomerular epithelial cells grown in chemically defined media, tubular epithelial cell morphology depended on whether the cells were placed in fibronectin or laminin and on the age of the donor animal used for culture. Primary tubular cells grown in laminin formed colonies; cells grown from young animals were rounded, whereas cells grown from adult animals were flattened. Primary tubular cells grown in fibronectin were flattened regardless of age, but cells from young animals formed colonies while those from adult animals formed a monolayer. Despite these differences in gross morphology, scanning and transmission electron microscopy revealed similar ultrastructural features in primary tubular cells from young and adult animals grown in fibronectin or laminin. Quantitative adhesion studies demonstrated that secondary subcultured tubular cells adhered equally well to dimeric and multimeric forms of fibronectin, but not to laminin. Quantitative colony growth studies of subcultured secondary tubular cells showed that laminin supports colony formation of trypsinized tubular cells, while previous work has demonstrated that fibronectin supports colony formation of glomerular cells. These results are consistent with the hypothesis that different extracellular matrix molecules are involved in colony formation of different cell types, with fibronectin stimulating growth of glomerular cells and laminin supporting growth of tubular cells.     

Epithelial tube morphogenesis during Drosophila tracheal development requires Piopio,a luminal ZP protein

The formation of branched epithelial networks is fundamental to the development of many organs, such as the lung, the kidney or the vasculature. Little is known about the mechanisms that control cell rearrangements during tubulogenesis and regulate the size of individual tubes. Recent studies indicate that whereas the basal surface of tube cells interacts with the surrounding tissues and helps to shape the ramification pattern of tubular organs, the apical surface has an important role in the regulation of tube diameter and tube growth. Here we report that two proteins, Piopio (Pio) and Dumpy (Dp), containing a zona pellucida (ZP) domain are essential for the generation of the interconnected tracheal network in Drosophila melanogaster. Pio is secreted apically, accumulates in the tracheal lumen and possibly interacts with Dp through the ZP domains. In the absence of Pio and Dp, multicellular tubes do not rearrange through cell elongation and cell intercalation to form narrow tubes with autocellular junctions; instead they are transformed into multicellular cysts, which leads to a severe disruption of the branched pattern. We propose that an extracellular matrix containing Pio and Dp provides a structural network in the luminal space, around which cell rearrangements can take place in an ordered fashion without losing interconnections. Our results suggest that a similar structural role might be attributed to other ZP-domain proteins in the formation of different branched organs.     

In Vitro Inhibition of Angiogenesis by Antibodies Directed against the 37kDa/67kDa Laminin Receptor

The 37kDa/67kDa laminin receptor (LRP/LR) is a central receptor mediating interactions between tumour cells and the basement membrane and is thereby a key player in adhesion and invasion, essential processes in metastatic cancer. To affect continued tumour growth, tumours induce angiogenesis for the constant delivery of nutrients and oxygen. This study aims to determine the blocking effect of the anti-LRP/LR specific antibody, W3 on the angiogenic potential of HUVE (human umbilical vein endothelial) cells. Flow cytometric analysis revealed that 97% of HUVE cells display cell surface LRP/LR. An angiogenesis assay was conducted employing HUVE cells seeded on the basement membrane reconstituent Matrigel™ supplemented with the pro-angiogenic factor vascular endothelial growth factor (VEGF). Post 18h incubation at 37°C tubular structures, namely tube lengths were assessed. Treatment of established tubular structures with 100 µg/ml anti-LRP/LR specific antibody completely blocked angiogenesis. Our findings suggest a central role of the 37kDa/67kDa LRP/LR in tube formation and recommends anti-LRP/LR specific antibodies as potential therapeutic tools for treatment of tumour angiogenesis.     

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.     

Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three- dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFalpha induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFalpha were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFalpha, suggesting that TNFalpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u- PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFalpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u- PAR by 72 and 46%, but TNFalpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFalpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFalpha is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.     

A model system for tumor angiogenesis: involvement of transforming growth factor-alpha in tube formation of human microvascular endothelial cells induced by esophageal cancer cells.

Tumor growth is dependent on angiogenesis, which is thought to be mediated through growth factors, such as transforming growth factor-alpha (TGF-alpha) and -beta (TGF-beta), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), produced by tumor cells. We have developed a model system for tumor angiogenesis in vitro: tube formation of human omentum microvascular endothelial (HOME) cells in type I collagen gels when these cells are co-cultured with tumor cells. Exogenously added TGF-alpha induced tube formation of HOME cells in collagen gel. In contrast, TGF-beta inhibited the TGF-alpha-induced tube formation of endothelial cells. We investigated whether tube formation could be induced in HOME cells in collagen gel when the HOME cells were co-cultured with three esophageal cancer cell lines, TE1, TE2, and TE5. TE1 and TE2 cells expressed both TGF-alpha and TGF-beta mRNA, but the level of TGF-alpha mRNA in TE2 was found to be much lower than in TE1 cells. TE5 did not express either TGF-alpha or TGF-beta. The tube formation of HOME cell was induced when they were co-cultured with TE1 cells, while both TE2 and TE5 cell lines induced tube formation at much lower rates than TE1. TE1-induced tube formation of HOME cells was specifically blocked by co-administration of anti-TGF-alpha-antibody, but not by anti-bFGF-antibody. The present study suggests that, in our model system, esophageal tumor angiogenesis is partly controlled by TGF-alpha, possibly through a paracrine pathway.     

Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens

Migration and tube formation of endothelial cells are important in angiogenesis and require a coordinated response to the extra-cellular matrix (ECM) and growth factor. Since focal adhesion kinase (FAK) integrates signals from both ECM and growth factor, we investigated its role in angiogenesis. Type I and II collagens are fibril-forming collagens and stimulate human umbilical vein endothelial cells (HUVECs) to form tube structure. Although knockdown of FAK restrained cell motility and resulted in inhibition of tube formation, FAK degradation and tube formation occurred simultaneously after incubation with fibril-forming collagens. The compensation for the FAK degradation by a calpain inhibitor or transient over-expression of FAK resulted in disturbance of tube formation. These phenomena are specific to fibril-forming collagens and mediated via α2β1 integrin. In conclusion, our data indicate that FAK is functioning in cell migration, but fibril-forming collagen-induced FAK degradation is necessary for endothelial tube formation.     

Geometrical microfeature cues for directing tubulogenesis of endothelial cells

Angiogenesis, the formation of new blood vessels by sprouting from pre-existing ones, is critical for the establishment and maintenance of complex tissues. Angiogenesis is usually triggered by soluble growth factors such as VEGF. However, geometrical cues also play an important role in this process. Here we report the induction of angiogenesis solely by SVVYGLR peptide micropatterning on polymer surfaces. SVVYGLR peptide stripes were micropatterned onto polymer surfaces by photolithography to study their effects on endothelial cell (EC) behavior. Our results showed that the EC behaviors (cell spreading, orientation and migration) were significantly more guided and regulated on narrower SVVYGLR micropatterns (10 and 50 μm) than on larger stripes (100 μm). Also, EC morphogenesis into tube formation was switched on onto the smaller patterns. We illustrated that the central lumen of tubular structures can be formed by only one-to-four cells due to geometrical constraints on the micropatterns which mediated cell-substrate adhesion and generated a correct maturation of adherens junctions. In addition, sprouting of ECs and vascular networks were also induced by geometrical cues on surfaces micropatterned with SVVYGLR peptides. These micropatterned surfaces provide opportunities for mimicking angiogenesis by peptide modification instead of exogenous growth factors. The organization of ECs into tubular structures and the induction of sprouting angiogenesis are important towards the fabrication of vascularized tissues, and this work has great potential applications in tissue engineering and tissue regeneration.     

斑马鱼管腔器官空腔形态发生及分子机制

后生动物复杂的体内结构和器官结构多以网络状的管道系统出现。中空的管腔作为这个系统的重要结构单元承担了运输物质、区分器官不同部位功能、分隔机体和外环境等诸多重要的生理功能。管腔的发育障碍将致使相关器官形态发生畸形、功能紊乱。管腔型器官形态发生易被直接观察以及各种相关突变鱼和荧光转基因鱼的出现, 使得斑马鱼(Danio rerio)成为管道器官研究的优秀模式动物。斑马鱼血管、神经管、小肠、胰腺外分泌腺、前肾管等几种重要的器官的形态发生都伴随着典型的腔道发育过程, 是研究管腔形成的重要器官模型。管腔形成由胞外信号诱导、细胞极性化、胞内物质定向运输、腔内液体形成和胞内细胞骨架重构等相关管腔细胞内外发生的结构功能变化过程所构成, 而这些结构与功能的变化过程是通过精确而复杂的分子调控网络来实现, 最终形成管道器官。文章对斑马鱼4种典型管腔型器官的空腔形态发生过程进行了综述, 并总结了此过程中的分子机制, 为今后的相关研究提供了参考。     

EGF Enhances ADSCs Secretion via ERK and JNK Pathways

The objective of this work was to study the effect of epidermal growth factor (EGF) induced secretions of angiogenesis factors in adipose-derived stem cells (ADSCs) and the involvement of mitogen-activated protein kinases (MAPK). ADSCs were cultured and ELISA assays were performed to quantify the vascular endothelial growth factor, the hepatocyte growth factor, and the stromal derived factor-1 in ADSC-conditioned medium before and after EGF treatments and after pharmacological inhibition of MAPKs with PD98059, SB203580, and SP600125. The tube formation assay was used to test the effects of EGF treated and inhibitor treated ADSCs on the human umbilical vein endothelial cells (HUVECs) tube formation. Liposuction was applied and ADSCs were cultured successfully. The ADSCs released a variety of angiogenic factors, with the EGF treatments enhancing secretions and promoting the HUVEC tube formation. The MAPK inhibitors PD98059 and SP600125 increased the paracrine to promote tubular formation, while the SB203580 played an opposite role. In conclusion, (1) the in vitro cultured ADSCs secrete various angiogenic factors and the EGF amplifies the secretion and can enhance the ADSCs on the HUVEC tube formation. (2) ERK1/2 and JNK pathway may be involved in the enhanced secretion capacity of ADSCs while the p38 pathway may exert an opposite effect.     

Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress.

Endothelial progenitor cells (EPCs), circulating in peripheral blood, migrate toward target tissue, differentiate, and contribute to the formation of new vessels. In this study, we report that shear stress generated by blood flow or tissue fluid flow can accelerate the proliferation, differentiation, and capillary-like tube formation of EPCs. When EPCs cultured from human peripheral blood were subjected to laminar shear stress, the cells elongated and oriented their long axes in the direction of flow. The cell density of the EPCs exposed to shear stress was higher, and a larger percentage of these cells were in the G2-M phase of the cell cycle, compared with EPCs cultured under static conditions. Shear stress markedly increased the EPC expression of two vascular endothelial growth factor receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase-1, and an intercellular adhesion molecule, vascular endothelial-cadherin, at both the protein and mRNA levels. Assays for tube formation in the collagen gels showed that the shear-stressed EPCs formed tubelike structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that EPCs are sensitive to shear stress and that their vasculogenic activities may be modulated by shear stress.     

Leukotriene C4 stimulates angiogenesis in bovine carotid artery endothelial cells in vitro

In order to investigate the mechanism of angiogenesis involved in inflammatory processes, the effects of leukotrienes and prostaglandin E2 on in vitro tube formation of cultured vascular endothelial cells were examined. Endothelial cells from bovine carotid artery were cultured for 4 days between two layers of collagen gel and the lengths of organized tubes were quantitatively estimated with an image analyzer. Treatment with 10(-8)-10(-6)M of prostaglandin E2 increased the tubular lengths, and leukotriene C4 stimulated tube formation at far lower concentrations (10(-15)-10(-9)M) but leukotriene B4 and D4 were not effective on the tube formation. It was also found that endothelial cell migration was stimulated by almost the same concentrations of leukotriene C4 as those stimulating tube formation. These data suggest that leukotriene C4 is, at least, one of the important factors involved in angiogenesis during inflammatory processes.