Modulation of Serine Proteinases and Metalloproteinases During Morphogenic Glial-Endothelial Interactions

Abstract: The regulation of microvessel formation and the expression of CNS-specific endothelial properties are attributed to perivascular astroglia. Specific proteolytic pathways mediate processes such as tissue remodeling, differentiation, invasion, and metastasis. We used a coculture system in which C6 glial cells induce CNS microvascular endothelial cells to form capillary-like structures to examine the role of plasminogen activators and collagenases in CNS microvessel morphogenesis. Fibrin zymography revealed the presence of high-molecular weight urokinase-type plasminogen activator (uPA), low-molecular weight uPA, and uPA/inhibitor complexes within endothelial cultures and cocultures. Gelatin zymography revealed the presence of 92-, 72-, and 62-kDa type IV collagenases within endothelial cultures and cocultures. uPA activity was confirmed by incubating the extracts with amiloride, an inhibitor of uPA. Collagenase activity was confirmed by incubating the gels with EDTA, an inhibitor of metalloproteinases. Quantitative densitometry showed a six- to eightfold decrease in coculture uPA during capillary-like structure formation. Substantially less change in type IV 72-kDa procollagenase activity was seen in cocultures during capillary-like structure formation, but active type IV 62-kDa collagenase activity was significantly increased during capillary-like structure formation. These findings establish that uPA and activated type IV collagenase activity specifically regulates morphogenic endothelial responses to glial interactions and suggest mechanisms by which microvessels respond within the CNS.     

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

Regulation of in vitro glia-induced microvessel morphogenesis by urokinase

Plasminogen activators (PAs) regulate a variety of processes involved in tissue morphogenesis and differentiation. We used a coculture system in which microvascular endothelial cells are induced by glial cells to form capillary-like structures in order to examine the role of urokinase-type PA (uPA) during microvessel morphogenesis within the central nervous system (CNS). Endotheliaderived uPA activity decreased sevenfold within glial-endothelial cocultures when capillary-like structures were formed. Incubation of cocultures with concentrations of phorbol 12-myristate 13-acetate (0.1 and 1.0 nM) that induced endothelial uPA activity (45–210%) inhibited endothelial differentiation (25–70%). Furthermore, incubation of cocultures with proteolytically active low molecular weight uPA (5–500 IU/ml) inhibited endothelial differentiation (37–75%), whereas the amino terminal cell-binding fragment of uPA had minimal effect. Inhibition of plasminogen activation in cocultures with the serine protease/plasmin inhibitors aprotinin and soybean trypsin inhibitor increased glia-induced capillary-like structure formation (96–98%). These findings establish a paracrine/autocrine function for urokinase and its inhibitors in regulating endothelial responses to perivascular glia and provide insight into mechanisms of microvascular reactions to CNS pathology. © 1994 Wiley-Liss, Inc.     

脑微血管内皮细胞条件培养液对星形胶质细胞的影响及通络中药的干预特征

目的:揭示脑微血管内皮细胞生理、病理及通络中药处理后不同状态的培养液对正常星形胶质细胞影响的特征,从细胞间相互作用角度探讨脑微血管内皮细胞与星形胶质细胞的生物学关系,为阐释脑微环境稳定的血脑屏障维护机制以及通络中药通过内皮细胞调节脑内微环境理论假说提供新的证据。方法:制备正常、拟缺血和拟缺血合并通络救脑注射液处理的大鼠脑微血管内皮细胞条件培养液,观察其对星形胶质细胞活性和凋亡率的影响。结果:与正常星形胶质细胞相比,正常内皮细胞条件培养液能够降低正常星形胶质细胞的活性,并促进星形胶质细胞的凋亡;而拟缺血处理的内皮细胞条件培养液能够提高正常星形胶质细胞的活性和凋亡率;拟缺血合并通络药物处理的内皮细胞条件培养液对正常星形胶质细胞的活性有提高作用,并显著降低其凋亡率。结论:三种不同处理方式的内皮细胞条件培养液对正常星形胶质细胞活性和凋亡产生不同的影响,提示不同状态的微血管内皮细胞对脑内微环境产生影响,通络救脑注射液可能通过调节微血管内皮细胞的分泌而对星形胶质细胞发挥作用。     

Activated microglia mediate neuronal cell injury via a nitric oxide mechanism.

Activated microglial have been proposed to play a pathogenetic role in immune-mediated neurodegenerative diseases. To test this hypothesis, purified murine neonatal microglial were cocultured with neuronal cells derived from fetal brain. Activation with IFN-gamma and LPS of these cocultures brought about a sharp decrease in uptake of gamma-amino butyric acid and a marked reduction in neuronal cell survival. These effects varied with the density of microglia, the concentrations of the activation signals (IFN-gamma and LPS), and the duration of coculture. Inasmuch as addition of NG-monomethyl-L-arginine blocked these effects, a L-arginine-dependent neurocytotoxic mechanism was implicated. Abundant nitrite, a metabolite of the free radical nitric oxide (NO) derived from L-arginine, was detected in activated microglial/neuronal cell cocultures and in purified microglial cell cultures but not in purified astrocyte or neuronal cell cultures, suggesting that microglial were the principal source of the NO. These findings support the hypothesis that microglia are the source of a neurocytotoxic-free radical, and shed light on an additional mechanism of immune-mediated brain injury.     

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.     

Target-derived astroglia regulate axonal outgrowth in a region-specific manner.

The potential neuroanatomical specificity of astrocyte influence on neurite outgrowth was studied using an in vitro coculture system in which neurons from embryonic rat spinal cord or hippocampus were grown for 4 days in the presence of, but not in direct contact with, astrocytes derived either from the same region (homotopic coculture) or from different regions (heterotopic coculture) of the rat central nervous system. The results showed that axonal outgrowth was greatly enhanced in heterotopic cocultures in which spinal cord or hippocampal neurons were grown with astrocytes derived from their appropriate CNS target regions. This effect was remarkably specific, because the astroglia harvested from spinal or hippocampal target regions were not effective in promoting axon growth of nonafferent neuronal populations. Dendritic outgrowth was similar under all coculture conditions. These data suggest that diffusible signals, produced by astrocytes, can regulate neurite extension in vitro in a neuroanatomically specific manner and that axons are more sensitive than dendrites to the regional astrocyte environment.     

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

Bone tissue engineering using adipose‐derived stem cells and endothelial cells: Effects of the cell ratio

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.     

Collapsin response mediator proteins of neonatal rat brain interact with chondroitin sulfate

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix.     

Secreted metalloproteinases in testicular cell culture

It is well known that cultured Sertoli cells secrete plasminogen activators (Lacroix et al., Mol Cell Endocrinol 1977; 9:227-236; Hettle et al., Biol Reprod 1986; 34:895-904). We now show that testicular cells in culture also secrete gelatinolytic metalloproteinases. Gelatin zymographic analysis of concentrated culture medium proteins reveals that Sertoli cells secrete gelatinases of 185 kDa, 110 kDa, 83 kDa, 76 kDa, and 72 kDa in addition to plasminogen activators (PAs). Gelatinase 185 kDa is induced by FSH. Media from Sertoli (epithelial)/peritubular (mesenchymal) cell cocultures contain the Sertoli cell gelatinases and one FSH-stimulated gelatinase of 50 kDa, indicating that gelatinase 50 kDa is regulated by both FSH and cell-cell interactions. A 50-kDa fibronectinolytic activity is also present in the coculture medium from cells grown in the presence of FSH. Casein zymography demonstrates a prominent 30-kDa protease only in media from cocultures. Peritubular cells secrete urokinase-type plasminogen activator (u-PA) and exhibit slight degrading activity at 86 kDa and 74 kDa. The gelatinases are most active in the pH range 7.3-8.5 and are completely or partially inhibited by metal ion chelators indicating that they are metalloproteinases. Our data demonstrate that testicular cells in culture secrete several gelatinases in addition to PAs, and that FSH and coculture conditions regulate some of these secreted proteases. We suggest that the highly regulated secretion of these proteases may well be of physiological importance during testicular development and spermatogenesis.     

Endothelial cell/macrophage cocultures as a model to study Streptococcus suis-induced inflammatory responses

Endothelial cells and macrophages are thought to play a critical role in the inflammatory response that contributes to meningitis. To investigate the interactions between Streptococcus suis and these two cell types, we developed a coculture model composed of brain microvascular endothelial cells and macrophage-like cells, and characterized the production of proinflammatory cytokines, chemokines, prostaglandin E₂ (PGE₂), and matrix metalloproteinase 9 (MMP-9) following a challenge with bacteria. Streptococcus suis cells stimulated the secretion of all the inflammatory mediators as well as MMP-9 in the coculture model. Responses to S. suis infection were influenced by endothelial cell/macrophage ratios and were dependent on the multiplicity of infection. Except for IL-6, significantly higher amounts of inflammatory mediators and MMP-9 were produced with the coculture model at an endothelial cell/macrophage ratio of 1 : 10 than at a ratio of 1 : 1. When infected with S. suis , endothelial cells and macrophages acted in synergy to increase the secretion of IL-6 and PGE₂. Using a model that more closely reproduces the in vivo situation, we showed that S. suis can induce the secretion of high levels of inflammatory mediators and MMP-9, which may contribute to the development of meningitis.     

Lipids in blood-brain barrier models in vitro II: Influence of glial cells on lipid classes and lipid fatty acids

Lipids of brain tissue and brain microvascular endothelial cells contain high proportions of long-chain polyunsaturated fatty acids (long PUFAs). The blood-brain barrier (BBB) is formed by the brain endothelial cells under the inductive influence of brain cells, especially perivascular glia, and coculture of endothelial cells and glial cells has been used to examine this induction. The objective of this study was to investigate whether C6 glioma cells are able to influence the lipid composition and shift the fatty acid (FA) patterns of the BBB model cell lines RBE4 and ECV304 toward the in vivo situation. Lipid classes of the three cell lines were analyzed by thin-layer chromatography and lipid FA patterns by high-performance liquid chromatography. Only ECV304 cells showed altered lipid composition in coculture with C6 cells. The fractions of triglycerides and cholesteryl esters (depending on the support filter) were about twice as high in coculture as when the cells were grown alone. Triglyceride fractions reached 13 to 15% of total lipids in coculture. The three cell lines showed an increase in the percentage of long PUFAs with respect to unsaturated FAs, mainly because of an increase in the percentages of arachidonic acid, all cis-7,10,13,16-docosatetraenoic acid, and all cis-7,10,13,16,19-docosapentaenoic acid. It is concluded that glioma C6 cells are able to induce a more in vivo-like FA pattern in BBB cell culture models. However, changes were not significant for the individual PUFAs, and their levels did not reach in vivo values.     

Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

Fibrin-enhanced endothelial cell organization

We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.     

Rapid communication Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

A new technique for primary hepatocyte expansion in vitro

The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.