Fluid shear stress and sphingosine 1-phosphate activate calpain to promote membrane type 1 matrix metalloproteinase (MT1-MMP) membrane translocation and endothelial invasion into three-dimensional collagen matrices

The vascular endothelium continually senses and responds to biochemical and mechanical stimuli to appropriately initiate angiogenesis. We have shown previously that fluid wall shear stress (WSS) and sphingosine 1-phosphate (S1P) cooperatively initiate the invasion of human umbilical vein endothelial cells into collagen matrices (Kang, H., Bayless, K. J., and Kaunas, R. (2008) Am. J. Physiol. Heart Circ. Physiol. 295, H2087-2097). Here, we investigated the role of calpains in the regulation of endothelial cell invasion in response to WSS and S1P. Calpain inhibition significantly decreased S1P- and WSS-induced invasion. Short hairpin RNA-mediated gene silencing demonstrated that calpain 1 and 2 were required for WSS and S1P-induced invasion. Also, S1P synergized with WSS to induce invasion and to activate calpains and promote calpain membrane localization. Calpain inhibition results in a cell morphology consistent with reduced matrix proteolysis. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown by others to regulate endothelial cell invasion, prompting us to test whether calpain acted upstream of MT1-MMP. S1P and WSS synergistically activated MT1-MMP and induced cell membrane localization of MT1-MMP in a calpain-dependent manner. Calpain activation, MT1-MMP activation and MT1-MMP membrane localization were all maximal with 5.3 dynes/cm(2) WSS and S1P treatment, which correlated with maximal invasion responses. Our data show for the first time that 5.3 dynes/cm(2) WSS in the presence of S1P combine to activate calpains, which direct MT1-MMP membrane localization to initiate endothelial sprouting into three-dimensional collagen matrices.     

Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices

Endothelial cell invasion is a key step in angiogenic blood vessel formation. Sphingosine-1-phosphate (S1P) has been previously reported to play a role in endothelial cell proliferation, survival, migration, and angiogenesis. Here, we examine the ability of S1P to regulate human endothelial cell invasion into three-dimensional collagen or fibrin matrices. We show that S1P potently stimulated human endothelial cell invasion, lumen formation, and branching morphogenesis in collagen, and fibrin matrices, (5- and 15-fold increases in invasion were observed, respectively). The S1P-induced invasion response was pertussis-toxin sensitive and completely dependent on integrins. Addition of integrin blocking reagents revealed that the alpha2beta1 integrin regulated invasion in collagen matrices, while a combination of alphavbeta3 and alpha5beta1 integrins regulated invasion in fibrin. Additionally, the S1P-induced invasion response was dependent on matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinase-3 (TIMP-3) was the only physiologic inhibitor of metalloproteinases that completely inhibited the potent stimulation of invasion induced by S1P. In contrast, TIMP-1 had no blocking effect on invasion or morphogenesis, while TIMP-2 and TIMP-4 partially reduced invasion but completely blocked lumen formation events. Collectively, these data reveal a marked ability of S1P to induce metalloproteinase- and integrin-dependent human endothelial cell invasion and morphogenesis in both collagen and fibrin three-dimensional matrices, the two most physiologically relevant matrices for angiogenesis.     

The effect of a native collagen gel substratum on the synthesis of collagen by bovine brain capillary endothelial cells

Cultured capillary endothelial cells, derived from bovine brain, and maintained on a plastic substratum synthesized predominantly interstitial collagens of which approximately 75 per cent were secreted into the medium. When grown on a native hydrated collagen type I gel, although no marked alteration in the 'collagen synthetic pattern' was observed, the overall level of collagen synthesis was increased by approximately 100 per cent. More dramatic, however, was the alteration in the distribution of these molecules between medium and cell layer. Interstitial collagens produced by cells grown on collagen gels were almost exclusively associated with the cell layer or collagenous gel. These studies, thus, demonstrate that an extracellular matrix may exert a considerable influence on the cellular synthetic activities and possibly cellular polarity of capillary endothelial cells.     

Toward single cell traction microscopy within 3D collagen matrices

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels.     

Second harmonic generation microscopy reveals altered collagen microstructure in usual interstitial pneumonia versus healthy lung

Background

It is not understood why some pulmonary fibroses such as cryptogenic organizing pneumonia (COP) respond well to treatment, while others like usual interstitial pneumonia (UIP) do not. Increased understanding of the structure and function of the matrix in this area is critical to improving our understanding of the biology of these diseases and developing novel therapies. The objectives herein are to provide new insights into the underlying collagen- and matrix-related biological mechanisms driving COP versus UIP.

Methods

Two-photon second harmonic generation (SHG) and excitation fluorescence microscopies were used to interrogate and quantify differences between intrinsic fibrillar collagen and elastin matrix signals in healthy, COP, and UIP lung.

Results

Collagen microstructure was different in UIP versus healthy lung, but not in COP versus healthy, as indicated by the ratio of forward-to-backward propagating SHG signal (F_SHG/B_SHG). This collagen microstructure as assessed by F_SHG/B_SHG was also different in areas with preserved alveolar architecture adjacent to UIP fibroblastic foci or honeycomb areas versus healthy lung. Fibrosis was evidenced by increased col1 and col3 content in COP and UIP versus healthy, with highest col1:col3 ratio in UIP. Evidence of elastin breakdown (i.e. reduced mature elastin fiber content), and increased collagen:mature elastin ratios, were seen in COP and UIP versus healthy.

Conclusions

Fibrillar collagen’s subresolution structure (i.e. “microstructure”) is altered in UIP versus COP and healthy lung, which may provide novel insights into the biological reasons why unlike COP, UIP is resistant to therapies, and demonstrates the ability of SHG microscopy to potentially distinguish treatable versus intractable pulmonary fibroses.     

Bi-directional cell contact-dependent regulation of gene expression between endothelial cells and osteoblasts in a three-dimensional spheroidal coculture model

Multiple cell-cell interactions control bone morphogenesis and vascularization. We have employed a spheroidal coculture system of endothelial cells (EC) and osteoblasts (OB) to study cell contact-dependent gene regulation between these two cell types that may play a role in regulating OB differentiation and EC angiogenic properties. Coculture spheroids differentiate spontaneously to organize into a core of OB and a surface layer of endothelial cells. Individual spheroid culture of EC or OB leads to significant alterations in gene expression compared to standard monolayer culture (upregulation of Tie-2 in EC; upregulation of angiopoietin-2 in osteoblasts). More importantly, spheroidal coculture of endothelial cells and osteoblasts leads to significant changes of gene expression in both cell populations (upregulation of VEGFR-2 in EC; downregulation of VEGF, and upregulation of alkaline phosphatase in osteoblasts). These changes are dependent on cell-cell contact and are not seen in stimulation experiments with conditioned supernatants. Collectively, the data demonstrate complex bi-directional gene regulation mechanisms between EC and OB that are likely to play a critical role during OB differentiation and in controlling the properties of angiogenic EC.     

Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation

Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.     

Behavior of endothelial cells on Matrigel and development of a method for a rapid and reproducible in vitro angiogenesis assay

During the process of angiogenesis, the normally quiescent endothelial cells that line the vasculature are induced to proliferate, migrate and align to form new blood vessels by angiogenic stimuli. Assays for angiogenic factors mostly involve in vivo approaches. The two most commonly used in vivo assays—the chick chorioallantoic membrane (CAM) assay and the rabbit corneal assay are tedious to perform and are technically demanding. Several in vitro assays have also been developed, based on the ability of endothelial cells to form tubes in 3-D matrices. Here, we describe the modification of a microcarrier bead-based assay. This assay combines cells grown on Cytodex-3 microcarrier beads with Matrigel to provide an easy, rapid, and reliable method for evaluating and measuring angiogenic activity. We also describe the differential behavior of normal and transformed endothelial cells cultured in Matrigel.     

A 3D in situ cell counter reveals that breast tumor cell (MDA‐MB‐231) proliferation rate is reduced by the collagen matrix density

Many cell types require the biophysical and biochemical cues within the 3D extracellular matrix (ECM) to exhibit their true physiologically relevant behavior. As a result, cell culture platforms have been evolving from traditional 2D petridish plates into 3D biomatrices, and there is a need for developing analytic tools to characterize 3D cell culture. The existing cell counting method, using a hemocytometer or coulter counter, requires that cells are suspended in fluids prior to counting. This poses a challenge for 3D cell culture as cells are embedded in a 3D biomatrix. We use a facile 3D cell counting method that overcomes this limitation and allows for in situ cell counting in a 3D cell culture using equipment that is commonly available in a biology lab. Using a breast tumor cell line, MDA‐MB‐231, as a model system, we demonstrated that MDA‐MB‐231 cells (1) grow slower within a 3D collagen matrix than on a 2D substrate for an extended growth time (a week) with a comparable, initial cell‐to‐cell distance, (2) their cell growth rate decreases with the increase of collagen concentration, showing a linear growth rate rather than an exponential growth rate. Further work using flow cytometry showed that the observed growth rate reduction was consistent with the retardation of the transition to S (synthesis) phase in the cell cycle. This work demonstrates the validity of the 3D cell counting method and the importance of cell–ECM interactions in cell proliferation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:990–996, 2015     

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.     

Inhibition of fibroblast-induced angiogenic phenotype of cultured endothelial cells by the overexpression of tissue inhibitor of metalloproteinase (TIMP)-3

In this study, we examined the effect of overexpression of tissue inhibitor of metalloproteinase (TIMP)-3 on the angiogenic phenotype expressed by vascular endothelial cells (ECs). ECs were infected with a recombinant adenovirus carrying the TIMP-3 gene at various multiplicities of infection, and TIMP-3 expression by transfected cells was confirmed by Western blotting and reverse zymography. At transfection doses of 6.25, 12.5, 25, 50 and 100 multiplicity of infection, EC migration was reduced to 66, 45, 25, 17 and 5%, respectively, of that of the control. At the multiplicity of infection of 20, capillary tube length was reduced by 80% compared to that of the control. Thus, expression of TIMP-3 by ECs effectively inhibited EC migration and tube formation. Overexpression of TIMP-3 by ECs may be considered a gene therapy strategy for the treatment of pathological angiogenesis such as cancer and diabetic retinopathy.     

ARHGEF4-mediates the actin cytoskeleton reorganization of hepatic stellate cells in 3-dimensional collagen matrices

The actin cytoskeleton of hepatic stellate cells (HSCs) is reorganized when they are cultured in 3D collagen matrices. Here, we investigated the molecular mechanism of actin cytoskeleton reorganization in HSCs cultured in 3D floating collagen matrices (FCM) compared to those on 2D polystyrene surfaces (PS). First, we found that the generation of dendritic cellular processes was controlled by Rac1. Next, we examined the differential gene expression of HSCs cultured on 2D PS and in 3D FCM by RNA-Seq and focused on the changes of actin cytoskeleton reorganization-related molecular components and guanine nucleotide exchange factors (GEFs). The results showed that the expression of genes associated with actin cytoskeleton reorganization-related cellular components, filopodia and lamellipodia, were significantly decreased, but podosome-related genes was significantly increased in 3D FCM. Furthermore, we found that a Rac1-specific GEF, ARHGEF4, played roles in morphological changes, migration and podosome-related gene expression in HSCs cultured in 3D FCM.

Abbreviations: 2D PS: 2-dimensional polystyrene surface; 3D FCM: 3-dimensional floating collagen matrices; ARHGEF4: Rho guanine nucleotide exchange factor 4; ARHGEF6: Rho guanine nucleotide exchange factor 6; GEF: guanine nucleotide exchange factor; HSC: hepatic stellate cell     

3D microenvironment of collagen hydrogel enhances the release of neurotrophic factors from human umbilical cord blood cells and stimulates the neurite outgrowth of human neural precursor cells

The umbilical cord blood (UCB) cells have been reported to secrete therapeutic signals, including a series of neurotrophic factors. This suggests the cell source provides suitable therapeutic environments for nerve regeneration that ultimately finds a possible cell therapy for nerve tissue. In this study, we observe a collagen hydrogel provides human UCB cells a proper 3D environment that stimulates the release of various neurotrophic factors. When compared to 2D culture, the 3D hydrogel culture significantly enhanced the expression of a series of neurotrophic factors, including neurotrophins, nerve growth factor, brain-derived neurotrophic factor, and ciliary neurotrophic factor as verified by the gene and protein analysis. To confirm the effects of neurotrophic factors secretion, we allowed an indirect interaction of the UCB-environment with human neural precursor cells (hNPCs). Results showed significantly enhanced neurite outgrowth of hNPCs. Collectively, our findings demonstrate that the collagen-based 3D hydrogel provides excellent environment for UCB-derived cells to release neurotrophic factors that will be ultimately useful for the neural repair and regeneration purposes.     

Heterogenous morphogenesis of Caco-2 cells reveals that flow induces three-dimensional growth and maturation at high initial seeding cell densities

Here, we introduce a customized hanging insert fitting a six-well plate to culture Caco-2 cells on hydrogel membranes under flow conditions. The cells are cultured in the apical channel-like chamber, which provides about 1.3 dyn/cm² shear, while the basolateral chamber is mixed when the device is rocked. The device was tested by investigating the functional impact of the initial seeding density in combination with flow applied at confluency. The low seeding density cultures grew in two dimensional (2D) irrespective of the flow. Flow and higher seeding density resulted in a mixture of three dimensional (3D) structures and 2D layers. Static culture and high cell seeding density resulted in 2D layers. The flow increased the height and ZO-1 expression of cells in 2D layers, which correlated with an improved barrier function. Cultures with 3D structures had higher ZO-1 expression than 2D cultures, but this did not correlate with an increased barrier function. 2D monolayers in static and dynamic cultures had similar morphology and heterogeneity in the expression of Mucin-2 and Villin, while the 3D structures had generally higher expression of these markers. The result shows that the cell density and flow determine 3D growth and that the highest barrier function was obtained with low-density cultures and flow.     

Three-dimensional imaging and quantification of real-time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

The development of a minimally invasive, robust, and inexpensive technique that permits real-time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold-based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real-time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high-resolution in obtaining the time-course of intracellular calcium levels without compromising spatial and temporal resolution. Three-dimensional reconstruction and depth-coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications.     

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

Angiogenesis, or neovascularization, is tightly orchestrated by endogenous regulators that promote or inhibit the process. The fine-tuning of these pro- and anti-angiogenic elements (the angiogenic balance) helps establish the homeostasis in tissues, and any aberration leads to pathologic conditions. The type I thrombospondin repeats are a family of protein structural elements involved in the control of angiogenesis, and some proteins containing these repeats have been identified as negative regulators of angiogenesis. Here we identify a set of 11 novel, anti-angiogenic 18–20-amino acid peptides that are derived from proteins that belong to the CCN protein family and contain type I thrombospondin motifs. We have named these peptides spondinstatin-1, cyrostatin, connectostatin, nephroblastostatin, wispostatin-2, wispostatin-3, netrinstatin-5C, netrinstatin-5D, adamtsostatin-like-4, fibulostatin-6.1, and complestatin-C6 to reflect their origin. We have shown that these peptides inhibit proliferation and migration of human umbilical vein endothelial cells in vitro. By conducting a clustering analysis of the amino acid sequences using sequence similarity criteria and of the experimental results using a hierarchical clustering algorithm, we have demonstrated that there is an underlying correlation between the sequence and activity of the identified peptides. This combination of experimental and computational approaches introduces a novel systematic framework for studying peptide activity, identifying novel peptides with anti-angiogenic activity, and designing mimetic peptides with tailored properties.     

Presenilin-1 controls the growth and differentiation of endothelial progenitor cells through its beta-catenin-binding region

Presenilin-1 (PS1) is a gene responsible for the development of early-onset familial Alzheimer's disease. Targeted disruption of the PS1 gene in mice suggested that PS1 might be involved in angiogenesis. We have used an in vitro embryonic stem (ES) cell culture system to prepare endothelial progenitor cells (EPC) lacking PS1 and investigated the roles of PS1 in endothelial cell lineage. With this system, Flk-1+ E-cadherin- EPC were generated from PS1-deficient ES cells, and the EPC lacking PS1 as well as wild-type EPC grew to form VE-cadherin+ endothelial colonies supported by a layer of OP9 stromal cells. Although the endothelial colonies from PS1-deficient EPC showed morphology similar to those from wild-type EPC, the PS1-deficient EPC formed a large number of the colonies compared to wild-type EPC. The enhanced colony-forming ability of PS1-deficient EPC was attenuated by the inductions of wild-type human PS1. To differentiate multiple activities of PS1 for colony-forming ability, we used two types of human PS1 mutants: one (hPS1D257A) with the aspartate to alanine mutation at residue 257 that impairs the proteolytic activity of PS1, and the other (hPS1Deltacat) deleting amino acids 340-371 of the cytosolic loop sequence essential for beta-catenin binding. hPS1D257A showed activity to regulate the colony-forming ability of PS1-deficient EPC, while hPS1Deltacat failed to exhibit this activity. These results suggest that PS1 regulates the growth and differentiation of endothelial progenitor cells through its beta-catenin-binding region and that the defect of PS1 function in endothelial cell lineage could contribute to the induction of vascular pathology.     

The role of type V collagen fibril as an ECM that induces the motility of glomerular endothelial cells

Although type V collagen (Col V) is present in developing and mature connective tissues of glomeruli, its primary function has not been elucidated yet. The purpose of this study was to elucidate the role of Col V fibrils in glomerular cells. We isolated primary cells from porcine kidney and cultured them on Col V fibrils reconstructed from purified Col V molecules extracted from porcine cornea. Time-lapse observation showed that Col V fibrils induce dynamic movement of glomerular endothelial cells (GEC) by stimulating them to extend long filopodial protrusions and wide lamellipodia. Col V signaling mediated through β1 integrin activated phosphorylation of paxillin at tyrosine 118 (paxillin-pY118) and of focal adhesion kinase at tyrosine 861 (FAKpY861) at the cell periphery; a second Col V signal was mediated through neuroglycan 2 and activated FAKpY397. FAKpY861 was present in loose attachment points between Col V fibrils and GEC, allowing the cells to migrate easily. Activation of FAKpY397 induced incomplete focal adhesion at the centers of cells and caused cell movement. Therefore both signaling pathways facilitated cell motility, which was inhibited by the addition of antibodies to β1 integrin, NG2, and Col V. We suggest that Col V fibrils activate ‘outside-in’ signaling in GEC and induce their dynamic motility.