Single-Cell Transcriptome Analysis of the Circle of Willis in a Mouse Cerebral Aneurysm Model.

BACKGROUND: The circle of Willis (CoW) is the most common location for aneurysms to form in humans. Although the major cell types of the intracranial vasculature are well known, the heterogeneity and relative contributions of the different cells in healthy and aneurysmal vessels have not been well characterized. Here, we present the first comprehensive analysis of the lineage heterogeneity and altered transcriptomic profiles of vascular cells from healthy and aneurysmal mouse CoW using single-cell RNA sequencing. METHODS: Cerebral aneurysms (CAs) were induced in adult male mice using an elastase model. Single-cell RNA sequencing was then performed on CoW samples obtained from animals that either had aneurysms form or rupture 14 days post-induction. Sham-operated animals served as controls. RESULTS: Unbiased clustering analysis of the transcriptional profiles from >3900 CoW cells identified 19 clusters representing ten cell lineages: vascular smooth muscle cells, endothelial cells fibroblasts, pericytes and immune cells (macrophages, T and B lymphocytes, dendritic cells, mast cells, and neutrophils). The 5 vascular smooth muscle cell subpopulations had distinct transcriptional profiles and were classified as proliferative, stress-induced senescent, quiescent, inflammatory-like, or hyperproliferative. The transcriptional signature of the metabolic pathways of ATP generation was found to be downregulated in 2 major vascular smooth muscle cell clusters when CA was induced. Aneurysm induction led to significant expansion of the total macrophage population, and this expansion was further increased with rupture. Both inflammatory and resolution-phase macrophages were identified, and a massive spike of neutrophils was seen with CA rupture. Additionally, the neutrophil-to-lymphocyte ratio (NLR), which originated from CA induction mirrored what happens in humans. CONCLUSIONS: Our data identify CA disease-relevant transcriptional signatures of vascular cells in the CoW and is searchable via a web-based R/shiny interface.

Hypothalamic Paraventricular Nucleus Gαi2 (Guanine Nucleotide-Binding Protein Alpha Inhibiting Activity Polypeptide 2) Protein-Mediated Neural Control of the Kidney and the Salt Sensitivity of Blood Pressure.

We have previously reported that in salt-resistant rat phenotypes brain, Gαi2 (guanine nucleotide-binding protein alpha inhibiting activity polypeptide 2) proteins are required to maintain blood pressure and sodium balance. However, the impact of hypothalamic paraventricular nucleus (PVN) Gαi2 proteins on the salt sensitivity of blood pressure is unknown. Here, by the bilateral PVN administration of a targeted Gαi2 oligodeoxynucleotide, we show that PVN-specific Gαi2 proteins are required to facilitate the full natriuretic response to an acute volume expansion (peak natriuresis [µeq/min] scrambled (SCR) oligodeoxynucleotide 41±3 versus Gαi2 oligodeoxynucleotide 18±4; P<0.05) via a renal nerve-dependent mechanism. Furthermore, in response to chronically elevated dietary sodium intake, PVN-specific Gαi2 proteins are essential to counter renal nerve-dependent salt-sensitive hypertension (mean arterial pressure [mm Hg] 8% NaCl; SCR oligodeoxynucleotide 128±2 versus Gαi2 oligodeoxynucleotide 147±3; P<0.05). This protective pathway involves activation of PVN Gαi2 signaling pathways, which mediate sympathoinhibition to the blood vessels and kidneys (renal norepinephrine [pg/mg] 8% NaCl; SCR oligodeoxynucleotide 375±39 versus Gαi2 oligodeoxynucleotide 850±27; P<0.05) and suppression of the activity of the sodium chloride cotransporter assessed as peak natriuresis to hydrochlorothiazide. Additionally, central oligodeoxynucleotide-mediated Gαi2 protein downregulation prevented PVN parvocellular neuron activation, assessed by FosB immunohistochemistry, in response to increased dietary salt intake. In our analysis of the UK BioBank data set, it was observed that 2 GNAI2 single nucleotide polymorphism (SNP) (rs2298952, P=0.041; rs4547694, P=0.017) significantly correlate with essential hypertension. Collectively, our data suggest that selective targeting and activation of PVN Gαi2 proteins is a novel therapeutic approach for the treatment of salt-sensitive hypertension.

Treatment with dimethyl fumarate reduces the formation and rupture of intracranial aneurysms: Role of Nrf2 activation.

Oxidative stress and chronic inflammation in arterial walls have been implicated in intracranial aneurysm (IA) formation and rupture. Dimethyl fumarate (DMF) exhibits immunomodulatory properties, partly via activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway which reduces oxidative stress by inducing the antioxidant response element (ARE). This study evaluated the effects of DMF both in vitro, using tumor necrosis factor (TNF)-α-treated vascular smooth muscle cells (VSMC), and in vivo, using a murine elastase model to induce aneurysm formation. The mice were treated with either DMF at 100 mg/kg/day P.O. or vehicle for two weeks. DMF treatment protected VSMCs from TNF-α-induced inflammation as demonstrated by its downregulation of cytokines and upregulation of Nrf2 and smooth muscle cell markers. At higher doses, DMF also inhibited the pro-proliferative action of TNF-α by increasing apoptosis which protected the cells from aponecrosis. In mice, DMF treatment significantly decreased the incidence of aneurysm formation and rupture, at the same time increasing Nrf2 levels. DMF demonstrated a neuroprotective effect in mice with a resultant inhibition of oxidative stress, inflammation, and fibrosis in the cerebrovasculature. This suggests a potential role for DMF as a rescue therapy for patients at risk for formation and rupture of IAs.

Cell Culture Model to Study Cerebral Aneurysm Biology.

Mechanisms governing cerebral aneurysm (CA) formation, progression, and rupture remain incompletely understood. However, understanding such mechanisms is critical to improving treatment for patients harboring CA. In vitro studies facilitate dissecting molecular mechanisms underlying vascular pathology and allow screening of therapies that can be subsequently explored in vivo. Cerebral vascular smooth muscle cells (VSMC) are an important constituent of the vessel wall, and phenotypic modulation of these cells to a pro-inflammatory, pro-matrix remodeling phenotype appears to be important in CA pathology. We have taken a reductionist approach using cultured cerebral VSMC to further explore CA biology. We describe techniques for culturing cerebral VSMC and outline experimental approaches incorporating these cells to study CA biology.

Cigarette Smoke Initiates Oxidative Stress-Induced Cellular Phenotypic Modulation Leading to Cerebral Aneurysm Pathogenesis.

OBJECTIVE: Cigarette smoke exposure (CSE) is a risk factor for cerebral aneurysm (CA) formation, but the molecular mechanisms are unclear. Although CSE is known to contribute to excess reactive oxygen species generation, the role of oxidative stress on vascular smooth muscle cell (VSMC) phenotypic modulation and pathogenesis of CAs is unknown. The goal of this study was to investigate whether CSE activates a NOX (NADPH oxidase)-dependent pathway leading to VSMC phenotypic modulation and CA formation and rupture. APPROACH AND RESULTS: In cultured cerebral VSMCs, CSE increased expression of NOX1 and reactive oxygen species which preceded upregulation of proinflammatory/matrix remodeling genes (MCP-1, MMPs [matrix metalloproteinase], TNF-α, IL-1ß, NF-κB, KLF4 [Kruppel-like factor 4]) and downregulation of contractile genes (SM-α-actin [smooth muscle α actin], SM-22α [smooth muscle 22α], SM-MHC [smooth muscle myosin heavy chain]) and myocardin. Inhibition of reactive oxygen species production and knockdown of NOX1 with siRNA or antisense decreased CSE-induced upregulation of NOX1 and inflammatory genes and downregulation of VSMC contractile genes and myocardin. p47phox-/- NOX knockout mice, or pretreatment with the NOX inhibitor, apocynin, significantly decreased CA formation and rupture compared with controls. NOX1 protein and mRNA expression were similar in p47phox-/- mice and those pretreated with apocynin but were elevated in unruptured and ruptured CAs. CSE increased CA formation and rupture, which was diminished with apocynin pretreatment. Similarly, NOX1 protein and mRNA and reactive oxygen species were elevated by CSE, and in unruptured and ruptured CAs. CONCLUSIONS: CSE initiates oxidative stress-induced phenotypic modulation of VSMCs and CA formation and rupture. These molecular changes implicate oxidative stress in the pathogenesis of CAs and may provide a potential target for future therapeutic strategies.

Lymphocytes influence intracranial aneurysm formation and rupture: role of extracellular matrix remodeling and phenotypic modulation of vascular smooth muscle cells.

BACKGROUND: Intracranial aneurysms (IA) are increasingly recognized as a disease driven by chronic inflammation. Recent research has identified key mediators and processes underlying IA pathogenesis, but mechanistic understanding remains incomplete. Lymphocytic infiltrates have been demonstrated in patient IA tissue specimens and have also been shown to play an important role in abdominal aortic aneurysms (AAA) and related diseases such as atherosclerosis. However, no study has systematically examined the contribution of lymphocytes in a model of IA. METHODS: Lymphocyte-deficient (Rag1) and wild-type (WT; C57BL/6 strain) mice were subjected to a robust IA induction protocol. Rates of IA formation and rupture were measured, and cerebral artery tissue was collected and utilized for histology and gene expression analysis. RESULTS: At 2 weeks, the Rag1 group had significantly fewer IA formations and ruptures than the WT group. Histological analysis of unruptured IA tissue showed robust B and T lymphocyte infiltration in the WT group, while there were no differences in macrophage infiltration, IA diameter, and wall thickness. Significant differences in interleukin-6 (IL-6), matrix metalloproteinases 2 (MMP2) and 9 (MMP9), and smooth muscle myosin heavy chain (MHC) were observed between the groups. CONCLUSIONS: Lymphocytes are key contributors to IA pathogenesis and provide a novel target for the prevention of IA progression and rupture in patients.

Kaolin-induced chronic hydrocephalus accelerates amyloid deposition and vascular disease in transgenic rats expressing high levels of human APP.

BACKGROUND: Normal pressure hydrocephalus (NPH) is most common in the elderly and has a high co-morbidity with Alzheimer's disease (AD) and cerebrovascular disease (CVD). To understand the relationship between NPH, AD and CVD, we investigated how chronic hydrocephalus impacts brain amyloid-beta peptide (Aß) accumulation and vascular pathology in an AD transgenic rodent model. Previously we showed that the altered CSF physiology produced by kaolin-hydrocephalus in older wild-type Sprague-Dawley rats increased Aß and hyperphosphorylated Tau (Silverberg et. al. Brain Res. 2010, 1317:286-296). We postulated that hydrocephalus would similarly affect an AD rat model. METHODS: Thirty-five transgenic rats (tgAPP21) that express high levels of human APP and naturally overproduce Aß40 were used. Six- (n = 7) and twelve-month-old (n = 9) rats had hydrocephalus induced by cisternal kaolin injection. We analyzed Aß burden (Aß40, Aß42 and oligomeric Aß) and vascular integrity (Masson trichrome and Verhoeff-Van Gieson) by immunohistochemistry and chemical staining at 10 weeks (n = 8) and 6 months (n = 5) post hydrocephalus induction. We also analyzed whether the vascular pathology seen in tgAPP21 rats, which develop amyloid angiopathy, was accelerated by hydrocephalus. Age-matched naïve and sham-operated tgAPP21 rats served as controls (n = 19). RESULTS: In hydrocephalic tgAPP21 rats, compared to naïve and sham-operated controls, there was increased Aß 40 and oligomeric Aß in hippocampal and cortical neurons at 10 weeks and 6 months post-hydrocephalus induction. No dense-core amyloid plaques were seen, but diffuse Aß immunoreactivity was evident in neurons. Vascular pathology was accelerated by the induction of hydrocephalus compared to controls. In the six-month-old rats, subtle degenerative changes were noted in vessel walls at 10 weeks post-kaolin, whereas at six months post-kaolin and in the 12-month-old hydrocephalic rats more pronounced amyloid angiopathic changes were seen, with frequent large areas of infarction noted. CONCLUSIONS: Kaolin-hydrocephalus can accelerate intraneuronal Aß40 accumulation and vascular pathology in tgAPP21 rats. In addition, disrupted CSF production and reduced CSF turnover results in impaired Aß clearance and accelerated vascular pathology in chronic hydrocephalus. The high co-morbidity seen in NPH, AD and CVD is likely not to be an age-related coincidence, but rather a convergence of pathologies related to diminished CSF clearance.

Gαi2-protein-mediated signal transduction: central nervous system molecular mechanism countering the development of sodium-dependent hypertension.

Excess dietary salt intake is an established cause of hypertension. At present, our understanding of the neuropathophysiology of salt-sensitive hypertension is limited by a lack of identification of the central nervous system mechanisms that modulate sympathetic outflow and blood pressure in response to dietary salt intake. We hypothesized that impairment of brain Gαi2-protein-gated signal transduction pathways would result in increased sympathetically mediated renal sodium retention, thus promoting the development of salt-sensitive hypertension. To test this hypothesis, naive or renal denervated Dahl salt-resistant and Dahl salt-sensitive (DSS) rats were assigned to receive a continuous intracerebroventricular control scrambled or a targeted Gαi2-oligodeoxynucleotide infusion, and naive Brown Norway and 8-congenic DSS rats were fed a 21-day normal or high-salt diet. High salt intake did not alter blood pressure, suppressed plasma norepinephrine, and evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi2-protein levels in naive Brown Norway, Dahl salt-resistant, and scrambled oligodeoxynucleotide-infused Dahl salt-resistant but not DSS rats. In Dahl salt-resistant rats, Gαi2 downregulation evoked rapid renal nerve-dependent hypertension, sodium retention, and sympathoexcitation. In DSS rats, Gαi2 downregulation exacerbated salt-sensitive hypertension via a renal nerve-dependent mechanism. Congenic-8 DSS rats exhibited sodium-evoked paraventricular nucleus-specific Gαi2-protein upregulation and attenuated hypertension, sodium retention, and global sympathoexcitation compared with DSS rats. These data demonstrate that paraventricular nucleus Gαi2-protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal nerve-dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Impairment of this mechanism contributes to the development of salt-sensitive hypertension.

Brain Gαi2-subunit protein-gated pathways are required to mediate the centrally evoked sympathoinhibitory mechanisms activated to maintain sodium homeostasis.

OBJECTIVE: We have previously demonstrated a role of GPCR-activated brain Gαi(2)-subunit protein-gated pathways in the natriuretic responses evoked by exogenous central α(2)-adrenoceptor activation and acute intravenous (i.v.) volume expansion in vivo. Our objective was to examine the role of brain Gαi(2) proteins in the integrated neural-humoral responses evoked by i.v. isovolumetric sodium loading, which does not alter mean arterial blood pressure or total blood volume, to maintain sodium homeostasis in conscious Sprague-Dawley rats. METHODS: Intact or chronic bilateral renal denervated (RDNX) rats were pretreated intracerebroventricularly (i.c.v.) with a scrambled or Gαi(2) oligodeoxynucleotide to selectively downregulate brain Gαi(2) proteins. On the day of study, an i.v. isovolumetric sodium load (1 mol/l NaCl) was administered. RESULTS: In naive and scrambled oligodeoxynucleotide groups, i.v. sodium loading evoked profound natriuresis, suppression of plasma renin activity (PRA) and global sympathoinhibition. Prior downregulation of brain Gαi(2) proteins significantly attenuated the natriuretic response [peak ΔUNaV (µeq/µl); scrambled 22 ± 2 vs. Gαi(2) 13 ± 2, P < 0.05] and abolished the sympathoinhibitory response [peak Δplasma norepinephrine (% control); SCR -72 ± 8 vs. Gαi(2) -7 ± 5, P < 0.05] without attenuating PRA suppression to sodium loading. In RDNX rats, Gαi(2) oligodeoxynucleotide pretreatment failed to attenuate the natriuretic response [peak ΔUNaV (µeq/µl); RDNX and scrambled 19 ± 3 vs. RDNX and Gαi(2) 20 ± 2] and only partially prevented the sympathoinhibitory response to i.v. sodium loading. CONCLUSION: These studies reveal a brain Gαi(2)-subunit protein-mediated (renin-angiotensin system-independent) sympathoinhibitory pathway that has a critical role in the central neural mechanisms activated to maintain fluid and electrolyte homeostasis.

Central nervous system Gαi2-subunit proteins maintain salt resistance via a renal nerve-dependent sympathoinhibitory pathway.

In salt-resistant phenotypes, chronic elevated dietary sodium intake evokes suppression of renal sodium-retaining mechanisms to maintain sodium homeostasis and normotension. We have recently shown that brain Gαi(2) protein pathways are required to suppress renal sympathetic nerve activity and facilitate maximal sodium excretion during acute intravenous volume expansion in Sprague-Dawley rats. Here, we studied the role of brain Gαi(2) proteins in the endogenous central neural mechanisms acting to maintain fluid and electrolyte homeostasis and normotension during a chronic elevation in dietary salt intake. Naive or bilaterally renal denervated adult male Sprague-Dawley rats were randomly assigned to receive an intracerebroventricular scrambled or Gαi(2) oligodeoxynucleotide infusion and then subjected to either a normal salt (0.4%) or high-salt (8.0%) diet for 21 days. In scrambled oligodeoxynucleotide-infused rats, salt loading, which did not alter blood pressure, evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi(2) protein levels and suppression of circulating norepinephrine content and plasma renin activity. In salt-loaded rats continuously infused intracerebroventricularly with a Gαi(2) oligodeoxynucleotide, animals exhibited sodium and water retention, elevated plasma norepinephrine levels, and hypertension, despite suppression of plasma renin activity. Furthermore, in salt-loaded bilaterally renal denervated rats, Gαi(2) oligodeoxynucleotide infusion failed to evoke salt-sensitive hypertension. Therefore, in salt-resistant rats subjected to a chronic high-salt diet, brain Gαi(2) proteins are required to inhibit central sympathetic outflow to the kidneys and maintain sodium balance and normotension. In conclusion, these data demonstrate a central role of endogenous brain, likely paraventricular nucleus-specific, Gαi(2)-subunit protein-gated signal transduction pathways in maintaining a salt-resistant phenotype.

Brain heterotrimeric Gαi2-subunit protein-gated pathways mediate central sympathoinhibition to maintain fluid and electrolyte homeostasis during stress.

Fluid and electrolyte homeostasis is integral to blood pressure regulation. However, the central molecular mechanisms regulating the neural control of sodium excretion remain unclear. We have demonstrated that brain Gαi(2)-subunit protein pathways mediate the natriuretic response to α(2)-adrenoreceptor activation in vivo. Consequently, we examined the role of brain Gαi(2) proteins in the neural mechanisms facilitating fluid and electrolyte homeostasis in response to acute [i.v. volume expansion (VE)] or chronic stressful stimuli (dietary sodium restriction vs. supplementation) in conscious Sprague-Dawley rats. Selective oligodeoxynucleotide (ODN)-mediated down-regulation of brain Gαi(2) proteins, but not a scrambled ODN, abolished the renal sympathoinhibitory response and attenuated the natriuresis to VE. In scrambled ODN-treated rats, chronic changes in dietary sodium intake evoked an endogenous, hypothalamic paraventricular nucleus (PVN)-specific, decrease (sodium deficiency) or increase (sodium excess) in PVN Gαi(2) proteins; plasma norepinephrine levels were inversely related to dietary sodium content. Finally, in rats treated with an ODN to prevent high salt-induced up-regulation of brain Gαi(2) proteins, animals exhibited sodium retention, global sympathoexcitation, and elevated blood pressure. Collectively, these data demonstrate that PVN Gαi(2) protein pathways play an endogenous role in maintaining fluid and electrolyte balance by controlling the influence the sympathetic nervous system has on the renal handling of sodium.

Amyloid-beta transporter expression at the blood-CSF barrier is age-dependent.

BACKGROUND: Age is the major risk factor for many neurodegenerative diseases, including Alzheimer's disease (AD). There is an accumulation of amyloid-beta peptides (Aß) in both the AD brain and the normal aging brain. Clearance of Aß from the brain occurs via active transport at the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB). With increasing age, the expression of the Aß efflux transporters is decreased and the Aß influx transporter expression is increased at the BBB, adding to the amyloid burden in the brain. Expression of the Aß transporters at the choroid plexus (CP) epithelium as a function of aging was the subject of this study. METHODS: This project investigated the changes in expression of the Aß transporters, the low density lipoprotein receptor-related protein-1 (LRP-1), P-glycoprotein (P-gp), LRP-2 (megalin) and the receptor for advanced glycation end-products (RAGE) at the BCSFB in Brown-Norway/Fischer rats at ages 3, 6, 9, 12, 20, 30 and 36 months, using real time RT-PCR to measure transporter mRNA expression, and immunohistochemistry (IHC) to measure transporter protein in isolated rat CP. RESULTS: There was an increase in the transcription of the Aß efflux transporters, LRP-1 and P-gp, no change in RAGE expression and a decrease in LRP-2, the CP epithelium influx transporter, at the BCSFB with aging. Decreased Aß42 concentration in the CP, as measured by quantitative IHC, was associated with these Aß transporter alterations. CONCLUSIONS: Age-dependent alterations in the CP Aß transporters are associated with a decrease in Aß42 accumulation in the CP, and are reciprocal to the changes seen in these transporters at the BBB, suggesting a possible compensatory role for the BCSFB in Aß clearance in aging.

Amyloid and Tau accumulate in the brains of aged hydrocephalic rats.

AD pathology is often seen in cortical biopsies of NPH patients. It remains unclear whether these findings are coincidental or causally related. In an aged animal model of NPH, we quantify Abeta and pTau accumulation and describe its temporal and spatial distribution. One-year-old male Sprague-Dawley rats had hydrocephalus induced by cisternal kaolin injection. Immunohistochemistry (IMHC) for AbetaPP, Abeta40, Abeta42 and pTau (epitope pT231) and ELISA for Abeta40, Abeta42 and pT231 were performed on controls and after 2, 6 and 10 weeks of hydrocephalus. Rats had double-label fluorescence IMHC for localization of Abeta42 and pT231. IMHC showed no change in neuronal AbetaPP expression following hydrocephalus. Abeta42 appeared earliest in CSF clearance pathways, p<0.05, and also showed significant rises in perivascular spaces and in cortical parenchyma. Mean ELISA values for Abeta40 and Abeta42 increased three- to four-fold in hydrocephalic rats at 6 and 10 weeks. Abeta40 increased between 2 and 6 weeks (p=0.0001), and remained stable at 10 (p=0.0002); whereas Abeta42 was elevated at 2 weeks (p<0.04) and remained at 6 (p=0.015). PTau at 6 and 10 weeks showed AD-like increased neuronal somatic staining and loss of dendritic staining. ELISA demonstrated increased pT231 in hydrocephalic rats at 10 weeks (p<0.0002). Double-label fluorescence for Abeta42 and pT231 revealed intraneuronal co-localization. Hydrocephalus in the elderly rat, therefore, can induce both Abeta and pTau accumulation. As distinct from brain injury models, no increase in AbetaPP expression was demonstrated. Rather, altered CSF dynamics appears to impair Abeta clearance in this NPH model.

Expression of junctional proteins in choroid plexus epithelial cell lines: a comparative study.

BACKGROUND: There is an increasing interest in using choroid plexus (CP) epithelial cell lines to study the properties of the blood-cerebrospinal fluid barrier (BCSFB). Currently, there are three major CP-derived cell lines available. Z310 and TR-CSFB3, two immortalized cell lines carrying the simian virus 40 large T-antigen gene, were derived from rat CP epithelium, whereas the CPC-2 cell line was derived from human CP carcinoma. Although these cell lines have previously been used in various functional studies, the expression of adherens junction (AJ) and tight junction (TJ) proteins in these epithelial cells has not been systematically studied. Accordingly, in the present study, we sought to characterize the expression of these junctional proteins in these three cell lines. METHODS: The cells were grown in six-well cell culture plates. Reverse-transcriptase polymerase chain reaction, Western blotting, and immunocytochemistry were used to characterize the expression of AJ and TJ proteins in the CP cell lines. RESULTS: Z310 and TR-CSFB3 cells expressed a TJ protein, occludin, and its cytosolic binding partner, zonula occludens 1, as well as an AJ protein, E-cadherin, and beta-catenin, a cytoplasmic protein that interacts with E-cadherin. However, the expression of occludin and E-cadherin in TR-CSFB3 cells at both the mRNA and protein level was weaker than that found in Z301 cells. The immunocytochemical analysis also demonstrated that the staining pattern for these junctional proteins in TR-CSFB3 cells was discontinuous and the staining intensity was weaker than that observed in Z310 cells. The message for claudin 1 and claudin 2 was expressed at low levels in TR-CSFB3 cells and these cells were weakly immunopositive for claudin 1. In comparison, the message for these TJ proteins could not be detected in Z310 cells. CPC-2 cells expressed occludin, which was localized to areas of cell-cell contact, but the staining pattern for this TJ protein was found to be variable and irregular. Although CPC-2 cells expressed mRNA for claudin 1, claudin 2, and claudin 11, only claudin 1 was expressed at the protein level and it was localized to the nuclei rather than to areas of cell-cell contact. An AJ protein, E-cadherin, was also found to be mislocalized in CPC-2 cells, even though its cytosolic binding partner, beta-catenin, was restricted to areas of cell-cell contact, as in normal CP. CONCLUSION: The three CP cell lines analyzed in this study vary considerably with regard to the expression of AJ and TJ proteins, which is likely reflected by different barrier properties of these in vitro models of BCSFB.