Breast adenocarcinoma cells adhere stronger to brain pericytes than to endothelial cells.

Most of the malignancies detected within the brain parenchyma are of metastatic origin. As the brain lacks classical lymphatic circulation, the primary way for metastasis relies on hematogenous routes. Dissemination of metastatic cells to the brain implies attachment to the luminal surface of brain endothelial cells, transmigration through the vessel wall, and adhesion to the brain surface of the vasculature. During this process, tumor cells must interact with brain endothelial cells and later on with pericytes. Physical interaction between tumor cells and brain vascular cells might be crucial in the successful extravasation of metastatic cells through blood vessels and later in their survival within the brain environment. Therefore, we applied single-cell force spectroscopy to investigate the nanoscale adhesive properties of living breast adenocarcinoma cells to brain endothelial cells and pericytes. We found target cell type-dependent adhesion characteristics, i.e. increased adhesion of the tumor cells to pericytes in comparison to endothelial cells, which underlines the existence of metastatic potential-related nanomechanical differences relying partly on membrane tether dynamics. Varying adhesion strength of the tumor cells to different cell types of brain vessels presumably reflects the transitory adhesion to endothelial cells before extravasation and the long-lasting strong interaction with pericytes during survival and proliferation in the brain. Our results highlight the importance of specific mechanical interactions between tumor cells and host cells during metastasis formation.

Inflammasome activation in peritumoral astrocytes is a key player in breast cancer brain metastasis development.

Inflammasomes, primarily responsible for the activation of IL-1ß, have emerged as critical regulators of the tumor microenvironment. By using in vivo and in vitro brain metastasis models, as well as human samples to study the role of the NLRP3 inflammasome in triple-negative breast cancer (TNBC) brain metastases, we found NLRP3 inflammasome components and IL-1ß to be highly and specifically expressed in peritumoral astrocytes. Soluble factors from TNBC cells induced upregulation and activation of NLRP3 and IL-1ß in astrocytes, while astrocyte-derived mediators augmented the proliferation of metastatic cells. In addition, inhibition of NLRP3 inflammasome activity using MCC950 or dampening the downstream effect of IL-1ß prevented the proliferation increase in cancer cells. In vivo, MCC950 reduced IL-1ß expression in peritumoral astrocytes, as well as the levels of inflammasome components and active IL-1ß. Most importantly, significantly retarded growth of brain metastatic tumors was observed in mice treated with MCC950. Overall, astrocytes contribute to TNBC progression in the brain through activation of the NLRP3 inflammasome and consequent IL-1ß release. We conclude that pharmacological targeting of inflammasomes may become a novel strategy in controlling brain metastatic diseases.

Cerebral Pericytes and Endothelial Cells Communicate through Inflammasome-Dependent Signals.

By upregulation of cell adhesion molecules and secretion of proinflammatory cytokines, cells of the neurovascular unit, including pericytes and endothelial cells, actively participate in neuroinflammatory reactions. As previously shown, both cell types can activate inflammasomes, cerebral endothelial cells (CECs) through the canonical pathway, while pericytes only through the noncanonical pathway. Using complex in vitro models, we demonstrate here that the noncanonical inflammasome pathway can be induced in CECs as well, leading to a further increase in the secretion of active interleukin-1ß over that observed in response to activation of the canonical pathway. In parallel, a more pronounced disruption of tight junctions takes place. We also show that CECs respond to inflammatory stimuli coming from both the apical/blood and the basolateral/brain directions. As a result, CECs can detect factors secreted by pericytes in which the noncanonical inflammasome pathway is activated and respond with inflammatory activation and impairment of the barrier properties. In addition, upon sensing inflammatory signals, CECs release inflammatory factors toward both the blood and the brain sides. Consequently, CECs activate pericytes by upregulating their expression of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome-forming pattern recognition receptor. In conclusion, cerebral pericytes and endothelial cells mutually activate each other in inflammation.

Breast adenocarcinoma-derived exosomes lower first-contact de-adhesion strength of adenocarcinoma cells to brain endothelial layer.

Despite of advances in modern therapeutics, one of the most feared complications of cancer are brain metastases, which often cause life impairing profound neurological symptoms and premature death. Breast adenocarcinoma is among the leading "sources" of brain metastases. Since the central nervous system lacks a classical lymphatic circulation, invading metastatic cells can reach the brain parenchyma only through haematogenous routes and must breach the blood-brain barrier (BBB). The key step before the transmigration of metastatic cells through the highly regulated interface of the BBB is the establishment of firm adhesion between the tumor cell and the cerebral endothelial layer. Using atomic force microscopy, as a high resolution force spectrograph, direct measurements of intercellular interactions was performed between living adenocarcinoma cells and a confluent endothelial layer pre-treated with carcinoma cell-derived exosomes. By immobilization of a living adenocarcinoma cell to an atomic force microscope's cantilever, intercellular de-adhesions were directly measured by single cell force spectroscopy (SCFS) at quasi-physiological conditions. De-adhesion dynamics and strength was characterized by several different calculated parameters, involving aspects of both membrane and cell surface related factors. Our results indicate that de-adhesion strength was lower in case of exosome pre-treated endothelial cells as compared to non-treated controls. Breast adenocarcinoma-derived exosomes have direct effect on de-adhesion pattern of brain endothelium.

SZR-104, a Novel Kynurenic Acid Analogue with High Permeability through the Blood-Brain Barrier.

By being an antagonist of glutamate and other receptors, kynurenic acid serves as an endogenous neuroprotectant in several pathologies of the brain. Unfortunately, systemic administration of kynurenic acid is hindered by its low permeability through the blood-brain barrier. One possibility to overcome this problem is to use analogues with similar biological activity as kynurenic acid, but with an increased permeability through the blood-brain barrier. We synthesized six novel aminoalkylated amide derivatives of kynurenic acid, among which SZR-104 (N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide) proved to have the highest permeability through an in vitro blood-brain barrier model. In addition, permeability of SZR-104 was significantly higher than that of kynurenic acid, xanthurenic acid and 39B, a quinolone derivative/xanthurenic acid analogue. Since peripherally administered SZR-104 is able to inhibit epileptiform activity in the brain, we conclude that SZR-104 is a promising kynurenic acid analogue with good penetrability into the central nervous system.

Gerardiins A-L and Structurally Related Phenanthrenes from the Halophyte Plant Juncus gerardii and Their Cytotoxicity against Triple-Negative Breast Cancer Cells.

Species in the Juncaceae accumulate different types of secondary metabolites, among them phenanthrenes and 9,10-dihydrophenanthrenes in substantial amounts. These compounds have chemotaxonomic significance and also possess interesting pharmacological activities. The present study has focused on the isolation, structure determination, and pharmacological investigation of phenanthrenes from Juncus gerardii. Twenty-six compounds, including 23 phenanthrenes, have been isolated from a methanol extract of this plant. Twelve compounds, the phenanthrenes gerardiins A-L (1-12), were obtained as new natural products. Eleven phenanthrenes [effusol (13), dehydroeffusol (14), effususin A (15), compressin A, 7-hydroxy-2-methoxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene, juncusol, 2-hydroxy-7-hydroxymethyl-1-methyl-5-vinyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-5-formyl-1-methyl-9,10-dihydrophenanthrene, effususol A, 2,7-dihydroxy-5-hydroxymethyl-1-methyl-9,10-dihydrophenanthrene, and jinflexin C], 1-O-p-coumaroyl-3-O-feruloyl-glycerol, and the flavones apigenin and luteolin were isolated for the first time from this plant. The cytotoxicity of the 23 isolated phenanthrenes in both mouse (4T1) and human (MDA-MB-231) triple-negative breast cancer cells and in a nontumor (D3, human cerebral microvascular endothelial) cell line was tested using an MTT viability assay. The results obtained showed that the dimeric compounds gerardiins I (9), J (10), K (11), and L (12), derived biogenetically from effusol and dehydroeffusol, were cytotoxic to both tumor and nontumor cell lines, while the monomeric compounds exerted no or very low cytotoxicity. Impedance measurements were consistent with the results of the MTT assays performed.

Pericyte-secreted IGF2 promotes breast cancer brain metastasis formation.

Brain metastases are life-threatening complications of triple-negative breast cancer, melanoma, and a few other tumor types. Poor outcome of cerebral secondary tumors largely depends on the microenvironment formed by cells of the neurovascular unit, among which pericytes are the least characterized. By using in vivo and in vitro techniques and human samples, here we show that pericytes play crucial role in the development of metastatic brain tumors by directly influencing key steps of the development of the disease. Brain pericytes had a prompt chemoattractant effect on breast cancer cells and established direct contacts with them. By secreting high amounts of extracellular matrix proteins, pericytes enhanced adhesion of both melanoma and triple-negative cancer cells, which might be particularly important in the exclusive perivascular growth of these tumor cells. In addition, pericytes secreted insulin-like growth factor 2 (IGF2), which had a very significant pro-proliferative effect on mammary carcinoma, but not on melanoma cells. By inhibiting IGF2 signaling using silencing or picropodophyllin (PPP), we could block the proliferation-increasing effect of pericytes on breast cancer cells. Administration of PPP (a blood-brain barrier-permeable substance) significantly decreased the size of brain tumors in mice inoculated with triple-negative breast cancer cells. Taken together, our results indicate that brain pericytes have significant pro-metastatic features, especially in breast cancer. Our study underlines the importance of targeting pericytes and the IGF axis as potential strategies in brain metastatic diseases.

Flavonoid, stilbene and diarylheptanoid constituents of Persicaria maculosa Gray and cytotoxic activity of the isolated compounds.

Persicaria maculosa (Polygonaceae) has been used as edible and as medicinal plant since ancient times. As a result of multistep chromatographic purifications, chalcones [2'-hydroxy-3',4',6'-trimethoxychalcone (1), pashanone (2), pinostrobin chalcone (3)], flavanones [6-hydroxy-5,7-dimethoxyflavanone (4), pinostrobin (5), onysilin (6), 5-hydroxy-7,8-dimethoxyflavanone (7)], flavonol [3-O-methylgalangin (8)], stilbene [persilben (9)], diarylheptanoids [1,7-diphenylhept-4-en-3-one (10), dihydroyashabushiketol (12), yashabushidiol B (13)] and 3-oxo-α-ionol-glucoside (11) were isolated from P. maculosa. The present paper reports for the first time the occurrence of diarylheptanoid-type constituents in the family Polygonaceae. Cytotoxicity of 1-5, 7 and 9-11 on 4 T1 mouse triple negative breast cancer cells was assayed by MTT test. None of the tested compounds reduced the cell viability to less than 80% of the control. On non-tumorigenic D3 human brain endothelial cells the decrease of cell viability was observed in case of 1 and 2. Further impedance measurements on 4 T1 and D3 cells a concentration-dependent decrease in the cell index of both cell types was demonstrated for 1, while 2 proved to be toxic only on endothelial cells.

Response of the neurovascular unit to brain metastatic breast cancer cells.

Therapeutic resistance of cerebral secondary tumours largely depends on unique aspects linked to the neurovascular unit, especially cerebral endothelial cells and astrocytes. By using advanced microscopy techniques, here we explored novel mechanisms related to the neurovascular unit during extravasation and proliferation of triple negative breast cancer cells in the brain. Metastatic mammary carcinoma cells arrested and elongated within one hour in cerebral microvessels, but their number decreased by almost 80% in the first two days. Interestingly, malignant cells induced vasoconstriction and development of intraluminal endothelial plugs, which isolated invading cells from the circulation. During diapedesis - which usually took place on day four and five after inoculation of the tumour cells - continuity of cerebral endothelial tight junctions remained intact, indicating migration of cancer cells through the transcellular pathway. In addition, metastatic cells induced formation of multiluminal vessels and claudin-5-positive endothelial blebs. However, even severe endothelial blebbing could be reversed and the vessel morphology was restored shortly after the tumour cells completed transendothelial migration. Similar to neuro-inflammatory leukocytes, tumour cells migrated not only through the endothelial layer, but through the glia limitans perivascularis as well. Nevertheless, along with the growth of metastatic lesions by co-option of pre-existing capillaries, astrocytes and astrocyte end-feet were gradually expelled from the vessels to the border of the tumour. Taken together, we identified previously unknown mechanisms involved in the reaction of brain resident cells to invading breast cancer cells. Our results contribute to a better understanding of the complex cross-talk between tumour cells and host cells in the brain, which is essential for the identification of new therapeutic targets in this devastating disease.

Pituitary adenylate cyclase activating polypeptide acts against neovascularization in retinal pigment epithelial cells.

The purpose of this study was to determine whether pituitary adenylate cyclase activating polypeptide (PACAP) could influence the neovascularization processes in hyperosmotic and oxidative stress in retinal pigment epithelial cells. Hyperosmotic conditions and oxidative stress were induced by 200 mM sucrose and 250 µM hydrogen peroxide (H2 O2 ), respectively. Morphology and elasticity of adult retinal pigment epithelial (ARPE-19) cells were measured by atomic force microscopy, while the investigation of junctional molecules, such as occludin and ZO-1, was carried out using immunofluorescence. For cell viability measurement, the MTT test was used. The effect of PACAP on the key angiogenic factors, such as vascular endothelial growth factor, angiogenin, and endothelin-1, was measured by an angiogenesis array and flow cytometry. Hyperosmotic stress-induced reorganization of the cytoskeleton and impairment of the junctions decreased cell viability and upregulated several angiogenic factors. In oxidative stress, we found that opening of the junctions decreased viability and upregulated the expression of angiogenic factors. PACAP was shown to be protective in both conditions. Retinal pigment epithelium cells play an important role in several diseases, such as diabetic retinopathy and macular edema. Therefore, protecting retinal pigment epithelial (RPE) cells with PACAP could be a novel and potential treatment in these diseases.

Paracellular and transcellular migration of metastatic cells through the cerebral endothelium.

Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases. Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood-brain barrier. By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps. We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia-like membrane protrusions towards the tumour cells. Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route. On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells. When co-culturing melanoma cells with cerebral endothelial cells, we observed N-cadherin enrichment at melanoma-melanoma and melanoma-endothelial cell borders. However, for breast cancer cells N-cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo. Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.

Rho-Kinase Inhibition Ameliorates Dasatinib-Induced Endothelial Dysfunction and Pulmonary Hypertension.

The multi-kinase inhibitor dasatinib is used for treatment of imatinib-resistant chronic myeloid leukemia, but is prone to induce microvascular dysfunction. In lung this can manifest as capillary leakage with pleural effusion, pulmonary edema or even pulmonary arterial hypertension. To understand how dasatinib causes endothelial dysfunction we examined the effects of clinically relevant concentrations of dasatinib on both human pulmonary arterial macro- and microvascular endothelial cells (ECs). The effects of dasatinib was compared to imatinib and nilotinib, two other clinically used BCR/Abl kinase inhibitors that do not inhibit Src. Real three-dimensional morphology and high resolution stiffness mapping revealed softening of both macro- and microvascular ECs upon dasatinib treatment, which was not observed in response to imatinib. In a dose-dependent manner, dasatinib decreased transendothelial electrical resistance/impedance and caused a permeability increase as well as disruption of tight adherens junctions in both cell types. In isolated perfused and ventilated rat lungs, dasatinib increased mean pulmonary arterial pressure, which was accompanied by a gain in lung weight. The Rho-kinase inhibitor Y27632 partly reversed the dasatinib-induced changes in vitro and ex vivo, presumably by acting downstream of Src. Co-administration of the Rho-kinase inhibitor Y27632 completely blunted the increased pulmonary pressure in response to dasatinib. In conclusion, a dasatinib-induced permeability increase in human pulmonary arterial macro- and microvascular ECs might explain many of the adverse effects of dasatinib in patients. Rho-kinase inhibition might be suitable to ameliorate these effects.

De-adhesion dynamics of melanoma cells from brain endothelial layer.

Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers.

Foe or friend? Janus-faces of the neurovascular unit in the formation of brain metastases.

Despite the potential obstacle represented by the blood-brain barrier for extravasating malignant cells, metastases are more frequent than primary tumors in the central nervous system. Not only tightly interconnected endothelial cells can hinder metastasis formation, other cells of the brain microenvironment (like astrocytes and microglia) can also be very hostile, destroying the large majority of metastatic cells. However, malignant cells that are able to overcome these harmful mechanisms may benefit from the shielding and even support provided by cerebral endothelial cells, astrocytes and microglia, rendering the brain a sanctuary site against anti-tumor strategies. Thus, cells of the neurovascular unit have a Janus-faced attitude towards brain metastatic cells, being both destructive and protective. In this review, we present the main mechanisms of brain metastasis formation, including those involved in extravasation through the brain vasculature and survival in the cerebral environment.

Expression of pattern recognition receptors and activation of the non-canonical inflammasome pathway in brain pericytes.

Cerebral pericytes are mural cells embedded in the basement membrane of capillaries. Increasing evidence suggests that they play important role in controlling neurovascular functions, i.e. cerebral blood flow, angiogenesis and permeability of the blood-brain barrier. These cells can also influence neuroinflammation which is highly regulated by the innate immune system. Therefore, we systematically tested the pattern recognition receptor expression of brain pericytes. We detected expression of NOD1, NOD2, NLRC5, NLRP1-3, NLRP5, NLRP9, NLRP10 and NLRX mRNA in non-treated cells. Among the ten known human TLRs, TLR2, TLR4, TLR5, TLR6 and TLR10 were found to be expressed. Inflammatory mediators induced the expression of NLRA, NLRC4 and TLR9 and increased the levels of NOD2, TLR2, inflammasome-forming caspases and inflammasome-cleaved interleukins. Oxidative stress, on the other hand, upregulated expression of TLR10 and NLRP9. Activation of selected pattern recognition receptors can lead to inflammasome assembly and caspase-dependent secretion of IL-1ß. TNF-α and IFN-γ increased the levels of pro-IL-1ß and pro-caspase-1 proteins; however, no canonical activation of NLRP1, NLRP2, NLRP3 or NLRC4 inflammasomes could be observed in human brain vascular pericytes. On the other hand, we could demonstrate secretion of active IL-1ß in response to non-canonical inflammasome activation, i.e. intracellular LPS or infection with E. coli bacteria. Our in vitro results indicate that pericytes might have an important regulatory role in neuroinflammation.

Direct mapping of melanoma cell - endothelial cell interactions.

The most life-threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high-resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi-physiological conditions. Hereby, we present our latest results by using this melanoma-decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single-cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic-related processes.

Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study.

The blood-brain barrier (BBB) is the main interface controlling molecular and cellular traffic between the central nervous system (CNS) and the periphery. It consists of cerebral endothelial cells (CECs) interconnected by continuous tight junctions, and closely associated pericytes and astrocytes. Different parts of the CNS have diverse functions and structures and may be subject of different pathologies, in which the BBB is actively involved. It is largely unknown, however, what are the cellular and molecular differences of the BBB in different regions of the brain. Using in silico, in vitro, and ex vivo techniques we compared the expression of BBB-associated genes and proteins (i.e., markers of CECs, brain pericytes, and astrocytes) in the cortical grey matter and white matter. In silico human database analysis (obtained from recalculated data of the Allen Brain Atlas), qPCR, Western blot, and immunofluorescence studies on porcine and mouse brain tissue indicated an increased expression of glial fibrillary acidic protein in astrocytes in the white matter compared with the grey matter. We have also found increased expression of genes of the junctional complex of CECs (occludin, claudin-5, and α-catenin) in the white matter compared with the cerebral cortex. Accordingly, occludin, claudin-5, and α-catenin proteins showed increased expression in CECs of the white matter compared with endothelial cells of the cortical grey matter. In parallel, barrier properties of white matter CECs were superior as well. These differences might be important in the pathogenesis of diseases differently affecting distinct regions of the brain.

Elasto-mechanical properties of living cells.

The possibility to directly measure the elasticity of living cell has emerged only in the last few decades. In the present study the elastic properties of two cell lines were followed. Both types are widely used as cell barrier models (e.g. blood-brain barrier). During time resolved measurement of the living cell elasticity a continuous quasi-periodic oscillation of the elastic modulus was observed. Fast Fourier transformation of the signals revealed that a very limited number of three to five Fourier terms fitted the signal in the case of human cerebral endothelial cells. In the case of canine kidney epithelial cells more than 8 Fourier terms did not result a good fit. Calculating the correlation between nucleus and periphery of the signals revealed a higher correlation factor for the endothelial cells compared to the epithelial cells.

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K.

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.

Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

BACKGROUND: Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. METHODS: Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. RESULTS: The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer. CONCLUSIONS: The data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.