Ultrastructural Pathology, Early Online, 1–10, 2015 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2014.981327
ORIGINAL ARTICLE
Intercellular Communication by Extracellular Vesicles with Emphasis on the Roles of Cordocytes in the Human Brain. An Ultrastructural Study Viorel Pais, PhD1 and Emil Sebastian Pais, DDS, MS2
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Independent Researcher, Bucharest, Romania and 2Cedars-Sinai Medical Center, Los Angeles, CA, USA
ABSTRACT We describe in this work the presence of extracellular vesicles (EVs) along different cell types, especially cordocytes, in various clinical conditions of the human brain (atherothrombotic disease, cerebral tumors, hygroma durae matris, intracerebral cysts, Moyamoya disease and parenchymatous hematoma) using transmission electron microscopy (TEM). EVs, illustrated as exosomes and microvesicles, were causally related to cell-to-cell communication, and other vital functions of resident cells around the brain parenchyma, either around the cortical vessels or into the subarachnoid space and the reticular arachnoid. Our direct demonstration by TEM of these information transporters in all locations and situations where the cordocytes play coordinating and regulating roles, producing and delivering a significant number of EVs to their targets, remains to be better documented in future studies. This first study on this topic showed clearly that EVs can be important modulators of cell functions with roles in cell activation, differentiation, phenotypic change, cancer progression, from precursor/stem cells to tumoral phenotypes, because EVs are released en masse during key interactions and certain moments. Keywords: Cell-to-cell communication, cordocytes, exosomes, human brain, microvesicles, ultrastructure
hope of using this information as a possible source to explain physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases [3–11]. In the human brain, the cordocyte is a new form of interstitial cell, recently characterized and found ubiquitously from the pia mater to the choroid plexus, with regulatory roles in numerous cell events, especially at the interface between brain parenchyma and meninges [12–14]. In this work, we describe for the first time the presence of EVs in cell–cell communication, especially concerning the behavior of cordocytes.
This consensus is that cordocytes are capable of forming an extensive intercellular information transmission and executive system that may use electric currents, small molecules, exosomes and possibly electrical events within the cytoskeleton to modulate homeostasis, stem cell activity, tissue repair, anticancer activity and other complex functions in many organs [1]. Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micronsized vesicles, which may deliver bioactive cargoes that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as RNA or non-coding RNA, released from the cell of origin to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress or death [2]. Ongoing studies investigate the molecular machinery and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the
MATERIALS AND METHODS We investigated by transmission electron microscopy (TEM) vascular walls and perivascular zones around the cortical arteries in three cases with middle cerebral artery thrombosis, a case with Moyamoya disease,
Received 7 October 2014; Accepted 22 October 2014; Published online 6 January 2015 Correspondence: Emil Sebastian Pais, DDS, MS, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA. Tel: (+1)310-423-3277. E-mail: [email protected]
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outer cerebral cortex in a case with transitional meningioma, arachnoid mater in a case with intracerebral cyst, a case with hygroma durae matris, an arteriolar wall in a case with parenchymatous hematoma, a microtumor belonging to the parenchymatous hemangiopericytoma, two fibrous meningiomas and a glioblastoma. Multiple blocks were processed using standard procedures for conventional electron microscopy. The specimens were fixed in a solution of 2.5% buffered glutaraldehyde to a pH of 7.2–7.3 for four hours. They were later postfixed in a solution of 1% buffered osmium tetroxide for 1 h, dehydrated with increasing concentrations of alcohols in water and embedded in resin epoxy (Epon 812). The resin was polymerized to 70 C for 72 h. Multiple ultrathin sections of 70 nm thickness were cut with an ultramicrotome and mounted on specimen grids covered with plastic films. These sections were contrasted with 2% uranyl acetate solution, as well as Reynolds lead citrate solution. The specimens were then examined under a JEM 1200-EX (Jeol Ltd., Tokyo, Japan) transmission electron microscope. The electron photomicrographs were processed on a computer and converted into images using Digital Micrograph acquisition software (Gatan, Inc., Pleasanton, CA).
FIGURE 1. Cell-to-cell communication between a cordocyte and a smooth muscle cell in the arterial wall. Significantly, there are microvesicles (arrowheads) passing from the cordocyte to subplasmalemmal endocytotic areas of smooth muscle cell (arrow), suggesting a role of the cordocyte for the vascular wall cells; 25,000.
RESULTS We analyzed the presence of microvesicles released as exosomes from the endosomal compartment and as shedding vesicles from the cell surface in a variety of cell types of the human brain in different clinical conditions, emphasizing on the presence of EVs along the cordocytes with their potential implications in reciprocal interactions. Cordocytes are special interstitial cells with very long prolongations capable of transferring signals and cytosolic material between distant cells. These cells, demonstrating important regulatory roles in the human brain, were observed in different pericortical areas, such as cortical vessels, outer cerebral cortex, subarachnoid space, arachnoid mater, vascular walls in Moyamoya disease and parenchymatous hematoma and a few cerebral tumors.
Cordocytes-smooth muscle cells communication in the vascular wall of cortical arteries In the inner adventitial layer of the cortical arteries were observed face-to-face both cordocytes and smooth muscle cells (SMCs) separated by a few collagen fibers. Many microvesicles were seen closely to the plasma membrane of the SMC, released in line by cordocytes. These microvesicles are characterized by their moderate electron-dense contents, with granules resembling ribosomes. In certain areas of
the plasma membranes, vesicle trafficking seemed to be an unidirectional continuous process, from cordocyte to SMC. Moreover, the cytoplasmic compartment of the SMC contained these endocytosed transferred microvesicles (Figure 1). Numerous other vesicles were also observed between cordocytes localized closely to the adventitial layer. As a general rule, the distribution of microvesicles is preferentially oriented toward other cells and not to the empty perivascular space. Occasionally, different stages of vesicle formation and membrane release may be seen nearby cell membrane. Shedding microvesicles may be formed at the end of some cell prolongations and directed to recipient cells by means of these fine processes.
Occurrence of the cordocytic EVs in the molecular layer of the cerebral cortex Cordocytes are localized in the molecular layer (Layer I), horizontally positioned like the horizontal cells of Cajal. Around cordocytes, one can see microvesicles released by these cells in the extracellular space (Figure 2).
Microvesicular traffic in the arachnoid mater Intensive vesicular trafficking was noted into the arachnoid mater, where numerous microvesicles are formed at the end of cell prolongations. Most of these microvesicles have a granular, ribosomal-type Ultrastructural Pathology
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Intercellular communication and cordocytes
FIGURE 2. Shedding vesicles along a cordocyte localized in the molecular layer of the cerebral cortex (arrows); 20,000.
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FIGURE 4. Prolongation of the arachnoid cells showing numerous microvesicles, some of them located in the cytoplasmic compartment (arrow). It is also visible a multivesicular body containing numerous exosomes (arrowhead); 25,000.
FIGURE 3. Exosomes and microvesicles localized between two arachnoid cells suggesting an active information exchange. Arrows indicate shedding vesicles and arrowheads indicate exosomes; 25,000.
FIGURE 5. Numerous microvesicles in the intercellular space or information at the cell surface (arrows); 25,000.
appearance. In some instances, microvesicles with granular contents may coexist with small, electronlucent exosomes, suggesting a multifaceted information exchange between cells (Figure 3). Extracellular multivesicular bodies containing numerous small exosomes were present around the cell processes (Figure 4). Unlike the exosomes, there are vesicles with different size and contents, and their apparition and migration to cell periphery is easily observed
(Figure 5). Furthermore, the presence of vesicular structures, either exosomes or shedding vesicles, are almost permanently seen in the narrowed spaces between cells with different degrees of differentiation and reciprocal vesicular exchange. Releasing and vesicle entrance in the same cell is also easily observed. The exchange of vesicular material between cells is a characteristic and, perhaps, a continuous
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process in the arachnoid mater. Shedding vesicles may be transferred and endocytosed in significant number in the areas with different phenotypic cells. Constantly, microvesicles are seen in both intracellular and extracellular spaces. As a rule, throughout the cell, microvesicles appear to be heavily granulated, but different in size, while the exosomes appear uniform in size and translucent in aspect.
were seen in line between cordocytic processes and differentiating cells into the areas with closest cell apposition (Figure 7). In these paracrine conditions, the interconnected cells are stabilized by collagen fibers produced by themselves or the cellular processes come close to each other, facilitating vesicular
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Cordocytes-stem cell communication by means of EVs in the subarachnoid space Dynamic interactions of the cordocytes with other cell types were seen throughout the examined areas, including the subarachnoid spaces, where many cell events occur, the pia mater itself being cordocytic in nature. The long cordocytic prolongations often showed numerous microvesicles in formation and released like a row in the extracellular space (Figure 6a and b). In this space, long and thin processes of the well-differentiated cordocytes may coexist with precursor/stem cells of the cordocytic lineage – these cells were characterized by an ovoid nucleus with characteristic heterochromatin geometry and a little cytoplasm with developing organelles, such as rough endoplasmic reticulum (ER), polyribosomes and a cluster of mitochondria. Furthermore, these cells are separated by several collagen fibers, which may confer stabilization of the partners. Significantly, there are distinctive plasma membrane ectodomains indicating microvesicle formation, and microvesicles were found in the extracellular space as well. In addition, small exosomes and microvesicles
FIGURE 7. A cordocyte localized in the reticular arachnoid surrounded by filiform processes, and little extracellular collagen matrix. Significantly, there are microvesicles in both extracellular space and the intracellular endocytotic compartment (long arrows). Short arrow indicates a microvesicular body containing exosomes, and arrowheads indicate vesicular dissolution in the extracellular space; 25,000.
FIGURE 6. (a) Cordocyte prolongations showing microvesicles at the plasma membrane and releasing them in a row into the extracellular space (arrows); 40,000. (b) This image shows cordocyte endings releasing numerous microvesicular structures around them (arrows); 40,000. Ultrastructural Pathology
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Intercellular communication and cordocytes
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action. In the subarachnoid space, as well as in the reticular arachnoid, in both cellular bodies and characteristically long cordocytic processes, numerous microvesicles were found shedded near the plasmalemmas. These microvesicles might remain close to cell membranes due to either the collagenous mass or interspersed processes, which form a dense inhibitory network against diffusible nanoparticles.
produced by cordocytes and oligodendrocytes. Another stem cell from the other vascular segment showed both microvesicles and exosomes on the cell membrane and into a deep incisure as well.
EVs released by cordocytes in vicinity of precursor/stem cells located in the vascular niche, in Moyamoya disease
Various differentiating cells were seen in the arteriolar wall, among them being both immature and mature cordocytic phenotypes localized outside the SMCs. Significantly, numerous shedding vesicles were released from both cellular body and, especially, from the long prolongations in vicinity of different cell types, including SMCs, fibroblasts, macrophages and even other differentiating cells on the cordocytic lineage (Figure 10).
In the vascular walls of the vessels analyzed in a case with Moyamoya disease, we also found evidence of cell-to-cell communication by the presence of numerous EVs, released by cordocytes and transferred to the precursor/stem cells or delivered into the lumen from the endothelium. These undifferentiated cells were surrounded by long and thin cytoplasmic processes of the well-differentiated cordocytes. We identified a large number of microvesicles in both cell processes and the extracellular space, arranged directionally toward the recipient cells (Figure 8). All these vesicles are similar to each other and seem to be continuously formed and released only in the vicinity of recipient cells. Moreover, we found numerous EVs in the vicinity of the vascular walls, i.e. in the nervous tissue, both on the surface of the myelinated axons and around the basement membrane, which separated nervous tissue from vessels (Figure 9). These EVs, both exosomes and microvesicles, can be
FIGURE 8. This image shows a precursor/stem cell on the lineage of cordocytes and processes of well-differentiated cordocytes in vicinity, which release numerous microvesicles (arrows). Arrowhead indicates exosomes; 25,000. !
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Shedding vesicles released by cordocytes in the arteriolar wall in a case with parenchymatous hematoma
EVs released by tumoral cells in some cases with cerebral tumors These microvesicles, as vehicles of cell communication, were present in other cell types than illustrated above, being released by tumoral cells sometimes, en masse. In a case with a microtumor belonging to an intraparenchymatous hemangiopericytoma, we observed numerous shedding vesicles on the endothelial surface and around the tumoral cells (Figure 11). These microvesicles have a fine granular content and similar vesicles were seen in the fibrous
FIGURE 9. Extracellular vesicles around the thin cordocytic processes surrounding nervous tissue (arrowhead) and shedding vesicles on the myelinated axons (arrow); 30,000.
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FIGURE 10. Typical cordocyte with ovoid nucleus and dense cytoplasm showing numerous shedding vesicles, ready to be released in vicinity (arrow); 20,000.
FIGURE 12. Numerous and identical to nanoparticles exosomes between tumor cell processes, in a case with fibrous meningioma (arrow); 20,000.
FIGURE 11. Numerous, irregular, and voluminous shedding vesicles on the endothelial surface from a microtumor developed in the white matter (arrow). Arrowhead indicates a microvesicle endocytosed in the tumor cell; 25,000.
FIGURE 13. Microvesicles of different sizes (arrows) and exosomes (arrowheads) on the surface of the tumor cell, in a case with glioblastoma; 8000.
Ultrastructural aspects of the disturbed vesicular traffic in cordocytes meningioma, when these microvesicular structures coexisted with numerous small exosomes (Figure 12). Microvesicular structures with different sizes were also found, especially on the surface of the tumoral cells at the periphery of glioblastoma where the proliferated and infiltrated tumoral cells have been intermingled with myelinated axons (Figure 13).
Vesicular transport is a major cellular activity responsible for molecular traffic, based on the selective packaging of the intended cargo into vesicles to be released and stimulating the effector cell. The release of the cargo from the ER, its accumulation in the Golgi zone and then cisternal progression toward exocytosis Ultrastructural Pathology
Intercellular communication and cordocytes
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DISCUSSION
FIGURE 14. This image shows a portion of the cordocyte displaying exaggerated and dilated Golgi cisterns, which appear empty and some of them opened outside the cell (arrow); 30,000.
FIGURE 15. Exaggerated bidirectional vesicular transport into the cordocyte, which seems to be modified in the normal phenotype (i.e. a cell with small body and long and thin processes). Cell body is increased in volume, with thickened and shortened processes, and contains an increased number of endocytosed vesicles (arrows); 30,000.
depend on the precise molecular control system. Occasionally, the ER and Golgi complex appear much dilated and enormous vesicles are released, while the vesicular trafficking appears normal in the neighboring cells (Figure 14). Furthermore, an exaggerated intensification of exocytosis and endocytosis for this vesicular transport might change the cellular phenotype of the cordocytes, which appear abnormally thickened, with short processes (Figure 15). If the ultrastructural pattern of cordocytes is normal then, even in necrotic tissue, microvesicles appear temporarily normal in size, number and aspect. !
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The direct demonstration by TEM of the cell-to-cell communication focusing on the cordocytic phenotype revealed new data about the important contribution of the EVs in the human brain, in both normal or pathological cellular events. A significant number of EVs belonging to the microvesicles and exosomes were released by cordocytes in their magistral interaction and communication with other cell types in different anatomical locations and biochemical conditions. Along the cordocytes, which are impressive by their lengths, microvesicles are preponderant versus exosomes and emitted toward other similar cells or different cell types, suggesting their supervisory function in local interactions. These special interstitial cells permanently coordinate and control other cells in all surrounding events, from precursor/stem cells evolution to different transitional types during repair processes. Therefore, the dynamic behavior of cordocytes depends on their capacity to rapidly develop long cytoplasmic processes, which might reach in time different targets, either other cordocytes or other cells encountered in their control zone. The information exchange is bidirectional, but cordocytes initiate contacts and respond first to the microenvironmental requirements. From literature data, now we understand the importance of these microvesicular structures in many facets of normal and pathological processes in the human brain. Molecules contained in the EVs are proteins, bioactive lipids and nucleic acids including TGS101, CD9, CD63, membrane receptors, messenger RNAs (mRNA), microRNAs (miRNA) [15], DNA [16] and mitochondrial DNA in some gliomas [17]. Some authors have asked where the epigenetic cargoes contained within the exosomes come from, how they are packaged in the cell and how they make their way to their targets [18]. At present, we have many gaps in our molecular knowledge concerning these carriers of cordocytes, from their biogenesis and secretion to important biological functions. However, according to our direct observations under TEM in cases where vesicular transfer takes place from cordocyte to another cell, only a minimal distance between the two cellular membranes exists, and microvesicles are released in line until necessary. It is known the significance of mesenchymal paracrine effect, and now mesenchymal stem cell-derived EVs may provide novel therapeutic approaches. Researchers are investigating the role of EVs in phenotypic cancer transformation and tumor progression. The uptake of tumor-derived EVs by noncancerous cells can change their normal phenotype to cancerous [19]. Suppression of vesiculation could slow down tumor growth and the spread of metastases. Formation of EVs in tumor cells include the following: (a) budding of plasma membrane, (b) release of exosomes after
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fusion of multivesicular body with plasma membrane and (c) non-apoptotic blebbing. Therefore, EVs are key contributors to cancer where they play an integral role in cell–cell communication and transfer prooncogenic molecules to recipient cells thereby conferring a cancerous phenotype. In addition, EVs secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-b1 and EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. According to our observations, numerous aberrant shedding vesicles are released from the endothelial surface in the case with parenchymatous hemangiopericytoma, an aggressive tumor, developed post-laceration in the white matter. A mass of microvesicular structures were also present at the surface of tumoral cells in glioblastoma, especially at the peripheral zone, suggesting EVs implication in tumor progression. Significantly, in meningiomas, exosomes were present in the narrowed spaces between tumoral cells, and not at distance, suggesting both their retention close to the cells and efficient implication in tumoral cell evolution. The potential clinical application of exosomes as biomarkers and therapeutic tools will be better documented in future studies [20,21]. So far, it is well established that the secreted EVs, such as exosomes and shedding vesicles, are key players in cell-to-cell communication, in intercellular signaling, and in modification of the target cell phenotype transferring both mRNA and miRNA [22]. For the central nervous system (CNS), exosomes are also mediators of neurodegeneration, neuroprotection and therapeutics [23]. As we demonstrated, an active communication between cordocytes themselves and between cordocytes and other cells around the brain parenchyma is essential to assure and synchronize a diversity of pericortical functions with the brain activity. These ubiquitous cells, from the vascular walls to the cerebral ectocortex, merit much more attention in future studies, in vitro and in vivo. Additional information is necessary to complete our observations according to the presence of cordocytes in the molecular layer (layer I) of the cerebral cortex in the same horizontal position, like the horizontal cells of Cajal [24]. The horizontal cells of Cajal, or simply referred to as horizontal cells, are also spindle-shaped cells located only in the superficial layers of the cerebral cortex. They give rise to a dendrite and an axon, both of which remain in the superficial layers of the cortex. Identification of these two different horizontal cells using TEM is necessary because the horizontal cells of Cajal may be seen, even seldom, in the adult brain. The brain is now regarded as an immune specialized system and cordocytes are definitely part of it. Neurons, astrocytes, oligodendrocytes (which transfer exosomes to neurons), microglia and cordocytes, as
well as brain tumor cells release EVs, which represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and the mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance [25]. In addition, identification and proteomic profiling of exosomes in human cerebrospinal fluid (CSF) was reported [26]. On the other hand, the arachnoid cells lining the CSF pathway show intense cell–cell communication and pinocytotic activity. This high transcellular activity reflects active transport and secretion of certain molecules by arachnoid cells. We demonstrated that cordocytes, as mesenchymal elements, may be interspersed with both neuroepithelial elements in the cerebral cortex, and the arachnoid mater add a new dimension for the complex interrelations in both brain and meninges, these supervisory cells themselves producing plenty of EVs. Moreover, they could modify the arachnoid cell phenotype because a massive release of these microvesicular structures exists at this microanatomical level. These pleiotropic EVs can easily reach their targets and act efficiently in all locations and interactions due to the adaptive cordocytic abilities [27]. Our electron micrographs illustrate a clear interrelationship between cordocytes and SMCs in the vascular wall, including a special situation from the Moyamoya disease with two different, adjacent phenotypes, i.e. a precursor cell for cordocyte lineage, and respectively, for smooth muscle phenotype. It remains to be determined certain molecules for their epigenetic function or tissue repair, when even a stem cell may limit injury or coordinate repair through the release of soluble factors [28]. A portion of EVs, visualized in the extracellular space, seem to be in dissolution, and their contents easily diffused in the local microenvironment. It is known that the specificity in the delivery of molecular cargo is essential for cell functions and survival. After delivering their cargo, most vesicles disappear or return via the reverse direction that is easily demonstrated by electron microscopy. On the other hand, EVs are carriers of pathogen-associated and damage-associated molecular patterns, cytokines, autoantigens and tissue-degrading enzymes [29]. Extracellular proteases have been detected in exosomes and they may alter the makeup of the recipient cell’s surface, being involved in special in tumor biology [30]. A recently recognized distinct type of intercellular communication device is the tunneling nanotube, which might play a role in prion spreading, and involved in neurological diseases in general [31,32]. Thus, the cellular connections, either homo- or heterocellular communications are very heterogeneous in both structure and function. The roles of Ultrastructural Pathology
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Intercellular communication and cordocytes cordocytes in the cell’s world of the human brain remain to be completed in future studies. We demonstrated in this work the presence of EVs as information transporters between cells in multiple locations and situations around the brain parenchyma. Traveling vesicles are produced mainly by well-differentiated cordocytes according to their specific signals, delivering their molecular cargoes for cell functions and survival, either to other similar cells including precursor/stem cells on the cordocyte lineage or to another cell type, such as SMCs, fibroblasts, arachnoid cells, etc. These interstitial cells, by their peculiar properties and impressive lengths, cover large anatomical areas and regulate numerous cell events around the brain parenchyma. In addition, their occasional presence in the molecular layer of the cerebral cortex merit more attention in future studies for cell-to-cell communication and other unexpected roles. Thus, their ubiquitarian presence with significant EVs release and efficiency open new research of the human brain.
CONCLUSION In this study, we report ultrastructural observations concerning the presence of microvesicular structures released by different cell types, most notable by cordocytes in the human brain. For the first time in literature, we point out the occurrence of these information vehicles between cordocytes and other cells in the brain in different clinical conditions. These results reinforce our opinion regarding the important roles of cordocytes during intercellular communications by means of EVs that these cells release in the proximity of their targets. Further studies are needed to assess the contents of these microvesicles and their specific roles and, respectively, exosomes. Finally, these vesicular structures seem to possess unexpected roles that will be elucidated in the future.
ACKNOWLEDGEMENTS Authors are grateful to Professor Dr. Leon Danaila for providing the clinical cases.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The authors acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by NIH (1S10RR23057 to ZHZ) and CNSI at UCLA. !
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Ultrastructural Pathology
ORIGINAL ARTICLE
Intercellular Communication by Extracellular Vesicles with Emphasis on the Roles of Cordocytes in the Human Brain. An Ultrastructural Study Viorel Pais, PhD1 and Emil Sebastian Pais, DDS, MS2
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Independent Researcher, Bucharest, Romania and 2Cedars-Sinai Medical Center, Los Angeles, CA, USA
ABSTRACT We describe in this work the presence of extracellular vesicles (EVs) along different cell types, especially cordocytes, in various clinical conditions of the human brain (atherothrombotic disease, cerebral tumors, hygroma durae matris, intracerebral cysts, Moyamoya disease and parenchymatous hematoma) using transmission electron microscopy (TEM). EVs, illustrated as exosomes and microvesicles, were causally related to cell-to-cell communication, and other vital functions of resident cells around the brain parenchyma, either around the cortical vessels or into the subarachnoid space and the reticular arachnoid. Our direct demonstration by TEM of these information transporters in all locations and situations where the cordocytes play coordinating and regulating roles, producing and delivering a significant number of EVs to their targets, remains to be better documented in future studies. This first study on this topic showed clearly that EVs can be important modulators of cell functions with roles in cell activation, differentiation, phenotypic change, cancer progression, from precursor/stem cells to tumoral phenotypes, because EVs are released en masse during key interactions and certain moments. Keywords: Cell-to-cell communication, cordocytes, exosomes, human brain, microvesicles, ultrastructure
hope of using this information as a possible source to explain physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases [3–11]. In the human brain, the cordocyte is a new form of interstitial cell, recently characterized and found ubiquitously from the pia mater to the choroid plexus, with regulatory roles in numerous cell events, especially at the interface between brain parenchyma and meninges [12–14]. In this work, we describe for the first time the presence of EVs in cell–cell communication, especially concerning the behavior of cordocytes.
This consensus is that cordocytes are capable of forming an extensive intercellular information transmission and executive system that may use electric currents, small molecules, exosomes and possibly electrical events within the cytoskeleton to modulate homeostasis, stem cell activity, tissue repair, anticancer activity and other complex functions in many organs [1]. Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micronsized vesicles, which may deliver bioactive cargoes that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as RNA or non-coding RNA, released from the cell of origin to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress or death [2]. Ongoing studies investigate the molecular machinery and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the
MATERIALS AND METHODS We investigated by transmission electron microscopy (TEM) vascular walls and perivascular zones around the cortical arteries in three cases with middle cerebral artery thrombosis, a case with Moyamoya disease,
Received 7 October 2014; Accepted 22 October 2014; Published online 6 January 2015 Correspondence: Emil Sebastian Pais, DDS, MS, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA. Tel: (+1)310-423-3277. E-mail: [email protected]
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outer cerebral cortex in a case with transitional meningioma, arachnoid mater in a case with intracerebral cyst, a case with hygroma durae matris, an arteriolar wall in a case with parenchymatous hematoma, a microtumor belonging to the parenchymatous hemangiopericytoma, two fibrous meningiomas and a glioblastoma. Multiple blocks were processed using standard procedures for conventional electron microscopy. The specimens were fixed in a solution of 2.5% buffered glutaraldehyde to a pH of 7.2–7.3 for four hours. They were later postfixed in a solution of 1% buffered osmium tetroxide for 1 h, dehydrated with increasing concentrations of alcohols in water and embedded in resin epoxy (Epon 812). The resin was polymerized to 70 C for 72 h. Multiple ultrathin sections of 70 nm thickness were cut with an ultramicrotome and mounted on specimen grids covered with plastic films. These sections were contrasted with 2% uranyl acetate solution, as well as Reynolds lead citrate solution. The specimens were then examined under a JEM 1200-EX (Jeol Ltd., Tokyo, Japan) transmission electron microscope. The electron photomicrographs were processed on a computer and converted into images using Digital Micrograph acquisition software (Gatan, Inc., Pleasanton, CA).
FIGURE 1. Cell-to-cell communication between a cordocyte and a smooth muscle cell in the arterial wall. Significantly, there are microvesicles (arrowheads) passing from the cordocyte to subplasmalemmal endocytotic areas of smooth muscle cell (arrow), suggesting a role of the cordocyte for the vascular wall cells; 25,000.
RESULTS We analyzed the presence of microvesicles released as exosomes from the endosomal compartment and as shedding vesicles from the cell surface in a variety of cell types of the human brain in different clinical conditions, emphasizing on the presence of EVs along the cordocytes with their potential implications in reciprocal interactions. Cordocytes are special interstitial cells with very long prolongations capable of transferring signals and cytosolic material between distant cells. These cells, demonstrating important regulatory roles in the human brain, were observed in different pericortical areas, such as cortical vessels, outer cerebral cortex, subarachnoid space, arachnoid mater, vascular walls in Moyamoya disease and parenchymatous hematoma and a few cerebral tumors.
Cordocytes-smooth muscle cells communication in the vascular wall of cortical arteries In the inner adventitial layer of the cortical arteries were observed face-to-face both cordocytes and smooth muscle cells (SMCs) separated by a few collagen fibers. Many microvesicles were seen closely to the plasma membrane of the SMC, released in line by cordocytes. These microvesicles are characterized by their moderate electron-dense contents, with granules resembling ribosomes. In certain areas of
the plasma membranes, vesicle trafficking seemed to be an unidirectional continuous process, from cordocyte to SMC. Moreover, the cytoplasmic compartment of the SMC contained these endocytosed transferred microvesicles (Figure 1). Numerous other vesicles were also observed between cordocytes localized closely to the adventitial layer. As a general rule, the distribution of microvesicles is preferentially oriented toward other cells and not to the empty perivascular space. Occasionally, different stages of vesicle formation and membrane release may be seen nearby cell membrane. Shedding microvesicles may be formed at the end of some cell prolongations and directed to recipient cells by means of these fine processes.
Occurrence of the cordocytic EVs in the molecular layer of the cerebral cortex Cordocytes are localized in the molecular layer (Layer I), horizontally positioned like the horizontal cells of Cajal. Around cordocytes, one can see microvesicles released by these cells in the extracellular space (Figure 2).
Microvesicular traffic in the arachnoid mater Intensive vesicular trafficking was noted into the arachnoid mater, where numerous microvesicles are formed at the end of cell prolongations. Most of these microvesicles have a granular, ribosomal-type Ultrastructural Pathology
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Intercellular communication and cordocytes
FIGURE 2. Shedding vesicles along a cordocyte localized in the molecular layer of the cerebral cortex (arrows); 20,000.
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FIGURE 4. Prolongation of the arachnoid cells showing numerous microvesicles, some of them located in the cytoplasmic compartment (arrow). It is also visible a multivesicular body containing numerous exosomes (arrowhead); 25,000.
FIGURE 3. Exosomes and microvesicles localized between two arachnoid cells suggesting an active information exchange. Arrows indicate shedding vesicles and arrowheads indicate exosomes; 25,000.
FIGURE 5. Numerous microvesicles in the intercellular space or information at the cell surface (arrows); 25,000.
appearance. In some instances, microvesicles with granular contents may coexist with small, electronlucent exosomes, suggesting a multifaceted information exchange between cells (Figure 3). Extracellular multivesicular bodies containing numerous small exosomes were present around the cell processes (Figure 4). Unlike the exosomes, there are vesicles with different size and contents, and their apparition and migration to cell periphery is easily observed
(Figure 5). Furthermore, the presence of vesicular structures, either exosomes or shedding vesicles, are almost permanently seen in the narrowed spaces between cells with different degrees of differentiation and reciprocal vesicular exchange. Releasing and vesicle entrance in the same cell is also easily observed. The exchange of vesicular material between cells is a characteristic and, perhaps, a continuous
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process in the arachnoid mater. Shedding vesicles may be transferred and endocytosed in significant number in the areas with different phenotypic cells. Constantly, microvesicles are seen in both intracellular and extracellular spaces. As a rule, throughout the cell, microvesicles appear to be heavily granulated, but different in size, while the exosomes appear uniform in size and translucent in aspect.
were seen in line between cordocytic processes and differentiating cells into the areas with closest cell apposition (Figure 7). In these paracrine conditions, the interconnected cells are stabilized by collagen fibers produced by themselves or the cellular processes come close to each other, facilitating vesicular
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Cordocytes-stem cell communication by means of EVs in the subarachnoid space Dynamic interactions of the cordocytes with other cell types were seen throughout the examined areas, including the subarachnoid spaces, where many cell events occur, the pia mater itself being cordocytic in nature. The long cordocytic prolongations often showed numerous microvesicles in formation and released like a row in the extracellular space (Figure 6a and b). In this space, long and thin processes of the well-differentiated cordocytes may coexist with precursor/stem cells of the cordocytic lineage – these cells were characterized by an ovoid nucleus with characteristic heterochromatin geometry and a little cytoplasm with developing organelles, such as rough endoplasmic reticulum (ER), polyribosomes and a cluster of mitochondria. Furthermore, these cells are separated by several collagen fibers, which may confer stabilization of the partners. Significantly, there are distinctive plasma membrane ectodomains indicating microvesicle formation, and microvesicles were found in the extracellular space as well. In addition, small exosomes and microvesicles
FIGURE 7. A cordocyte localized in the reticular arachnoid surrounded by filiform processes, and little extracellular collagen matrix. Significantly, there are microvesicles in both extracellular space and the intracellular endocytotic compartment (long arrows). Short arrow indicates a microvesicular body containing exosomes, and arrowheads indicate vesicular dissolution in the extracellular space; 25,000.
FIGURE 6. (a) Cordocyte prolongations showing microvesicles at the plasma membrane and releasing them in a row into the extracellular space (arrows); 40,000. (b) This image shows cordocyte endings releasing numerous microvesicular structures around them (arrows); 40,000. Ultrastructural Pathology
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action. In the subarachnoid space, as well as in the reticular arachnoid, in both cellular bodies and characteristically long cordocytic processes, numerous microvesicles were found shedded near the plasmalemmas. These microvesicles might remain close to cell membranes due to either the collagenous mass or interspersed processes, which form a dense inhibitory network against diffusible nanoparticles.
produced by cordocytes and oligodendrocytes. Another stem cell from the other vascular segment showed both microvesicles and exosomes on the cell membrane and into a deep incisure as well.
EVs released by cordocytes in vicinity of precursor/stem cells located in the vascular niche, in Moyamoya disease
Various differentiating cells were seen in the arteriolar wall, among them being both immature and mature cordocytic phenotypes localized outside the SMCs. Significantly, numerous shedding vesicles were released from both cellular body and, especially, from the long prolongations in vicinity of different cell types, including SMCs, fibroblasts, macrophages and even other differentiating cells on the cordocytic lineage (Figure 10).
In the vascular walls of the vessels analyzed in a case with Moyamoya disease, we also found evidence of cell-to-cell communication by the presence of numerous EVs, released by cordocytes and transferred to the precursor/stem cells or delivered into the lumen from the endothelium. These undifferentiated cells were surrounded by long and thin cytoplasmic processes of the well-differentiated cordocytes. We identified a large number of microvesicles in both cell processes and the extracellular space, arranged directionally toward the recipient cells (Figure 8). All these vesicles are similar to each other and seem to be continuously formed and released only in the vicinity of recipient cells. Moreover, we found numerous EVs in the vicinity of the vascular walls, i.e. in the nervous tissue, both on the surface of the myelinated axons and around the basement membrane, which separated nervous tissue from vessels (Figure 9). These EVs, both exosomes and microvesicles, can be
FIGURE 8. This image shows a precursor/stem cell on the lineage of cordocytes and processes of well-differentiated cordocytes in vicinity, which release numerous microvesicles (arrows). Arrowhead indicates exosomes; 25,000. !
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Shedding vesicles released by cordocytes in the arteriolar wall in a case with parenchymatous hematoma
EVs released by tumoral cells in some cases with cerebral tumors These microvesicles, as vehicles of cell communication, were present in other cell types than illustrated above, being released by tumoral cells sometimes, en masse. In a case with a microtumor belonging to an intraparenchymatous hemangiopericytoma, we observed numerous shedding vesicles on the endothelial surface and around the tumoral cells (Figure 11). These microvesicles have a fine granular content and similar vesicles were seen in the fibrous
FIGURE 9. Extracellular vesicles around the thin cordocytic processes surrounding nervous tissue (arrowhead) and shedding vesicles on the myelinated axons (arrow); 30,000.
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FIGURE 10. Typical cordocyte with ovoid nucleus and dense cytoplasm showing numerous shedding vesicles, ready to be released in vicinity (arrow); 20,000.
FIGURE 12. Numerous and identical to nanoparticles exosomes between tumor cell processes, in a case with fibrous meningioma (arrow); 20,000.
FIGURE 11. Numerous, irregular, and voluminous shedding vesicles on the endothelial surface from a microtumor developed in the white matter (arrow). Arrowhead indicates a microvesicle endocytosed in the tumor cell; 25,000.
FIGURE 13. Microvesicles of different sizes (arrows) and exosomes (arrowheads) on the surface of the tumor cell, in a case with glioblastoma; 8000.
Ultrastructural aspects of the disturbed vesicular traffic in cordocytes meningioma, when these microvesicular structures coexisted with numerous small exosomes (Figure 12). Microvesicular structures with different sizes were also found, especially on the surface of the tumoral cells at the periphery of glioblastoma where the proliferated and infiltrated tumoral cells have been intermingled with myelinated axons (Figure 13).
Vesicular transport is a major cellular activity responsible for molecular traffic, based on the selective packaging of the intended cargo into vesicles to be released and stimulating the effector cell. The release of the cargo from the ER, its accumulation in the Golgi zone and then cisternal progression toward exocytosis Ultrastructural Pathology
Intercellular communication and cordocytes
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DISCUSSION
FIGURE 14. This image shows a portion of the cordocyte displaying exaggerated and dilated Golgi cisterns, which appear empty and some of them opened outside the cell (arrow); 30,000.
FIGURE 15. Exaggerated bidirectional vesicular transport into the cordocyte, which seems to be modified in the normal phenotype (i.e. a cell with small body and long and thin processes). Cell body is increased in volume, with thickened and shortened processes, and contains an increased number of endocytosed vesicles (arrows); 30,000.
depend on the precise molecular control system. Occasionally, the ER and Golgi complex appear much dilated and enormous vesicles are released, while the vesicular trafficking appears normal in the neighboring cells (Figure 14). Furthermore, an exaggerated intensification of exocytosis and endocytosis for this vesicular transport might change the cellular phenotype of the cordocytes, which appear abnormally thickened, with short processes (Figure 15). If the ultrastructural pattern of cordocytes is normal then, even in necrotic tissue, microvesicles appear temporarily normal in size, number and aspect. !
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The direct demonstration by TEM of the cell-to-cell communication focusing on the cordocytic phenotype revealed new data about the important contribution of the EVs in the human brain, in both normal or pathological cellular events. A significant number of EVs belonging to the microvesicles and exosomes were released by cordocytes in their magistral interaction and communication with other cell types in different anatomical locations and biochemical conditions. Along the cordocytes, which are impressive by their lengths, microvesicles are preponderant versus exosomes and emitted toward other similar cells or different cell types, suggesting their supervisory function in local interactions. These special interstitial cells permanently coordinate and control other cells in all surrounding events, from precursor/stem cells evolution to different transitional types during repair processes. Therefore, the dynamic behavior of cordocytes depends on their capacity to rapidly develop long cytoplasmic processes, which might reach in time different targets, either other cordocytes or other cells encountered in their control zone. The information exchange is bidirectional, but cordocytes initiate contacts and respond first to the microenvironmental requirements. From literature data, now we understand the importance of these microvesicular structures in many facets of normal and pathological processes in the human brain. Molecules contained in the EVs are proteins, bioactive lipids and nucleic acids including TGS101, CD9, CD63, membrane receptors, messenger RNAs (mRNA), microRNAs (miRNA) [15], DNA [16] and mitochondrial DNA in some gliomas [17]. Some authors have asked where the epigenetic cargoes contained within the exosomes come from, how they are packaged in the cell and how they make their way to their targets [18]. At present, we have many gaps in our molecular knowledge concerning these carriers of cordocytes, from their biogenesis and secretion to important biological functions. However, according to our direct observations under TEM in cases where vesicular transfer takes place from cordocyte to another cell, only a minimal distance between the two cellular membranes exists, and microvesicles are released in line until necessary. It is known the significance of mesenchymal paracrine effect, and now mesenchymal stem cell-derived EVs may provide novel therapeutic approaches. Researchers are investigating the role of EVs in phenotypic cancer transformation and tumor progression. The uptake of tumor-derived EVs by noncancerous cells can change their normal phenotype to cancerous [19]. Suppression of vesiculation could slow down tumor growth and the spread of metastases. Formation of EVs in tumor cells include the following: (a) budding of plasma membrane, (b) release of exosomes after
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fusion of multivesicular body with plasma membrane and (c) non-apoptotic blebbing. Therefore, EVs are key contributors to cancer where they play an integral role in cell–cell communication and transfer prooncogenic molecules to recipient cells thereby conferring a cancerous phenotype. In addition, EVs secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-b1 and EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. According to our observations, numerous aberrant shedding vesicles are released from the endothelial surface in the case with parenchymatous hemangiopericytoma, an aggressive tumor, developed post-laceration in the white matter. A mass of microvesicular structures were also present at the surface of tumoral cells in glioblastoma, especially at the peripheral zone, suggesting EVs implication in tumor progression. Significantly, in meningiomas, exosomes were present in the narrowed spaces between tumoral cells, and not at distance, suggesting both their retention close to the cells and efficient implication in tumoral cell evolution. The potential clinical application of exosomes as biomarkers and therapeutic tools will be better documented in future studies [20,21]. So far, it is well established that the secreted EVs, such as exosomes and shedding vesicles, are key players in cell-to-cell communication, in intercellular signaling, and in modification of the target cell phenotype transferring both mRNA and miRNA [22]. For the central nervous system (CNS), exosomes are also mediators of neurodegeneration, neuroprotection and therapeutics [23]. As we demonstrated, an active communication between cordocytes themselves and between cordocytes and other cells around the brain parenchyma is essential to assure and synchronize a diversity of pericortical functions with the brain activity. These ubiquitous cells, from the vascular walls to the cerebral ectocortex, merit much more attention in future studies, in vitro and in vivo. Additional information is necessary to complete our observations according to the presence of cordocytes in the molecular layer (layer I) of the cerebral cortex in the same horizontal position, like the horizontal cells of Cajal [24]. The horizontal cells of Cajal, or simply referred to as horizontal cells, are also spindle-shaped cells located only in the superficial layers of the cerebral cortex. They give rise to a dendrite and an axon, both of which remain in the superficial layers of the cortex. Identification of these two different horizontal cells using TEM is necessary because the horizontal cells of Cajal may be seen, even seldom, in the adult brain. The brain is now regarded as an immune specialized system and cordocytes are definitely part of it. Neurons, astrocytes, oligodendrocytes (which transfer exosomes to neurons), microglia and cordocytes, as
well as brain tumor cells release EVs, which represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and the mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance [25]. In addition, identification and proteomic profiling of exosomes in human cerebrospinal fluid (CSF) was reported [26]. On the other hand, the arachnoid cells lining the CSF pathway show intense cell–cell communication and pinocytotic activity. This high transcellular activity reflects active transport and secretion of certain molecules by arachnoid cells. We demonstrated that cordocytes, as mesenchymal elements, may be interspersed with both neuroepithelial elements in the cerebral cortex, and the arachnoid mater add a new dimension for the complex interrelations in both brain and meninges, these supervisory cells themselves producing plenty of EVs. Moreover, they could modify the arachnoid cell phenotype because a massive release of these microvesicular structures exists at this microanatomical level. These pleiotropic EVs can easily reach their targets and act efficiently in all locations and interactions due to the adaptive cordocytic abilities [27]. Our electron micrographs illustrate a clear interrelationship between cordocytes and SMCs in the vascular wall, including a special situation from the Moyamoya disease with two different, adjacent phenotypes, i.e. a precursor cell for cordocyte lineage, and respectively, for smooth muscle phenotype. It remains to be determined certain molecules for their epigenetic function or tissue repair, when even a stem cell may limit injury or coordinate repair through the release of soluble factors [28]. A portion of EVs, visualized in the extracellular space, seem to be in dissolution, and their contents easily diffused in the local microenvironment. It is known that the specificity in the delivery of molecular cargo is essential for cell functions and survival. After delivering their cargo, most vesicles disappear or return via the reverse direction that is easily demonstrated by electron microscopy. On the other hand, EVs are carriers of pathogen-associated and damage-associated molecular patterns, cytokines, autoantigens and tissue-degrading enzymes [29]. Extracellular proteases have been detected in exosomes and they may alter the makeup of the recipient cell’s surface, being involved in special in tumor biology [30]. A recently recognized distinct type of intercellular communication device is the tunneling nanotube, which might play a role in prion spreading, and involved in neurological diseases in general [31,32]. Thus, the cellular connections, either homo- or heterocellular communications are very heterogeneous in both structure and function. The roles of Ultrastructural Pathology
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Intercellular communication and cordocytes cordocytes in the cell’s world of the human brain remain to be completed in future studies. We demonstrated in this work the presence of EVs as information transporters between cells in multiple locations and situations around the brain parenchyma. Traveling vesicles are produced mainly by well-differentiated cordocytes according to their specific signals, delivering their molecular cargoes for cell functions and survival, either to other similar cells including precursor/stem cells on the cordocyte lineage or to another cell type, such as SMCs, fibroblasts, arachnoid cells, etc. These interstitial cells, by their peculiar properties and impressive lengths, cover large anatomical areas and regulate numerous cell events around the brain parenchyma. In addition, their occasional presence in the molecular layer of the cerebral cortex merit more attention in future studies for cell-to-cell communication and other unexpected roles. Thus, their ubiquitarian presence with significant EVs release and efficiency open new research of the human brain.
CONCLUSION In this study, we report ultrastructural observations concerning the presence of microvesicular structures released by different cell types, most notable by cordocytes in the human brain. For the first time in literature, we point out the occurrence of these information vehicles between cordocytes and other cells in the brain in different clinical conditions. These results reinforce our opinion regarding the important roles of cordocytes during intercellular communications by means of EVs that these cells release in the proximity of their targets. Further studies are needed to assess the contents of these microvesicles and their specific roles and, respectively, exosomes. Finally, these vesicular structures seem to possess unexpected roles that will be elucidated in the future.
ACKNOWLEDGEMENTS Authors are grateful to Professor Dr. Leon Danaila for providing the clinical cases.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The authors acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by NIH (1S10RR23057 to ZHZ) and CNSI at UCLA. !
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