Molecular Changes Associated with the Protective Effects of Angiopoietin-1 During Blood-Brain Barrier Breakdown Post-Injury.

Blood-brain barrier (BBB) breakdown to plasma proteins leads to vasogenic edema which when diffuse is a life threatening complication in many types of acute brain injury. In our previous studies, early BBB breakdown was associated with increased expression of endothelial caveolin-1α (Cav-1) protein and decreased expression of occludin. In order to attenuate these changes, the effects of intra-cortical angiopoietin-1 (Ang1), a potent anti-permeability factor, on BBB breakdown was assessed in the cold injury model at day 1 post-injury. Overall vascular permeability at the lesion site was assessed in Ang1 non-treated and treated cold-injured rats, using horseradish peroxidase (HRP) as a tracer and in individual vessels by dual labeling immunofluorescence for Cav-1 or occludin and fibronectin, a marker of BBB breakdown. In addition, Cav-1, occludin, Akt, and ERK1/2 expression at the lesion site was detected by immunoblotting. Non-treated cold-injured rats showed focal HRP leakage at the lesion site which was significantly decreased (P < 0.001) in the Ang1-treated group. Increased endothelial Cav-1 and decreased occludin immunoreactivity was observed in arterioles and corresponding-sized venules with BBB breakdown in the non-treated cold-injured rats, and similar expression of these proteins was detected at the lesion site by immunoblotting associated with increased expression of Akt and ERK2 proteins. These alterations were attenuated by Ang1 treatment which resulted in Cav-1, occludin, Akt, and ERK1/2 protein expression that was similar to that of the control groups as was the endothelial Cav-1 and occludin immunoreactivity in leaky vessels. These data suggest that Ang1 administered early post-injury has potential in attenuating the degree of vascular alterations and subsequent vasogenic edema.

Feeder-independent derivation of induced-pluripotent stem cells from peripheral blood endothelial progenitor cells.

Induced-pluripotent stem cells (iPSCs) are a potential alternative cell source in regenerative medicine, which includes the use of differentiated iPSCs for cell therapies to treat coronary artery and/or peripheral arterial diseases. Late-outgrowth endothelial progenitor cells (late-EPCs) are a unique primary cell present in peripheral blood that exhibit high proliferative capacity, are being used in a wide variety of clinical trials, and have the ability to differentiate into mature endothelial cells. The objective of this study was to reprogram peripheral blood-derived late-EPCs to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs that were retrovirally transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs.

Deciphering the complexities of human diseases and disorders by coupling induced-pluripotent stem cells and systems genetics.

The recent discovery that adult mouse and human somatic cells can be 'reprogrammed' to a state of pluripotency by ectopic expression of only a few transcription factors has already made a major impact on the biomedical community. For the first time, it is possible to study diseases on an individual patient basis, which may eventually lead to the realization of personalized medicine. The utility of induced-pluripotent stem cells (iPSCs) for modeling human diseases has greatly benefitted from established human embryonic stem cell (ESC) differentiation and tissue engineering protocols developed to generate many different cell and tissue types. The limited access to preimplantation genetic tested embryos and the difficulty in gene targeting human ESCs have restricted the use of human ESCs in modeling human disease. Afforded by iPSC technology is the ability to study disease pathogenesis as it unfolds during tissue morphogenesis. The complexities of molecular signaling and interplay with protein transduction during disease progression necessitate a systems approach to studying human diseases, whereby data can be statistically integrated by sorting out the signal to noise issues that arise from global biological changes associated with disease versus experimental noise. Using a systems approach, biomarkers can be identified that define the initiation or progression of disease and likewise can serve as putative therapeutic targets.

PCL2 modulates gene regulatory networks controlling self-renewal and commitment in embryonic stem cells.

Recent reports have better elucidated the components of the Polycomb Repressive Complex 2 (PRC2) and its functional role in embryonic stem cells (ESCs) and their differentiated derivatives. The depletion of a newly described mammalian PRC2 associated protein, PCL2, leads to an increase in ESC self-renewal and a delay in differentiation, a phenotype similar to knockouts of the core PRC2 members. Genomic and cell biology data suggest that PCL2 is important in cell fate decisions and may play a role in recruitment of PRC2 to target genes and histone methylation. Importantly, depletion of PCL2 in ESCs leads to a decrease in 3meH3K27 at the proximal promoter regions of pluripotency transcription factors Tbx3, Klf4, Foxd3 and a concomitant increase in gene expression. These proteins subsequently activate expression of Oct4, Nanog and Sox2 through a feed-forward gene regulatory circuit, altering the core pluripotency network and driving cell fate decisions towards self-renewal. We propose a model whereby alteration of the epigenetic state of Tbx3, Klf4, and Foxd3 results in the enforced expression of the pluripotency network, preventing differentiation.

Detection of multiple proteins in intracerebral vessels by confocal microscopy.

Assessment of the blood-brain barrier (BBB) may involve the localization of endothelial proteins within the context of endothelial permeability to plasma proteins. The use of antibodies conjugated to fluorescent dyes, coupled with analysis by confocal microscopy, allows for the detection of multiple proteins in components of the neurovascular unit including endothelium and astrocytes. This chapter provides a detailed protocol for detection of three proteins in fixed or frozen sections of rat brain using three fluorophores with unique excitation/emission spectra. Also included is a protocol for tyramide signal amplification, which is useful for detecting proteins of low abundance, and methods for quantitation of intracerebral vessels expressing a particular protein of interest with and without BBB breakdown to plasma proteins.

Pathology and new players in the pathogenesis of brain edema.

Brain edema continues to be a major cause of mortality after diverse types of brain pathologies such as major cerebral infarcts, hemorrhages, trauma, infections and tumors. The classification of edema into vasogenic, cytotoxic, hydrocephalic and osmotic has stood the test of time although it is recognized that in most clinical situations there is a combination of different types of edema during the course of the disease. Basic information about the types of edema is provided for better understanding of the expression pattern of some of the newer molecules implicated in the pathogenesis of brain edema. These molecules include the aquaporins, matrix metalloproteinases and growth factors such as vascular endothelial growth factors A and B and the angiopoietins. The potential of these agents in the treatment of edema is discussed. Since many molecules are involved in the pathogenesis of brain edema, effective treatment cannot be achieved by a single agent but will require the administration of a "magic bullet" containing a variety of agents released at different times during the course of edema in order to be successful.

Fate of connexin43 in cardiac tissue harbouring a disease-linked connexin43 mutant.

AIMS: More than 40 mutations in the GJA1 gene encoding connexin43 (Cx43) have been linked to oculodentodigital dysplasia (ODDD), a pleiotropic, autosomal dominant disorder. We hypothesized that even with a significant reduction in the levels of Cx43 in a mutant mouse model of ODDD (Gja1(Jrt/+)) harbouring a G60S mutation (Cx43(G60S)), cardiomyocyte function may only be moderately compromised given that a majority of mutant mice typically survive. METHODS AND RESULTS: Western blotting and quantitative reverse transcriptase-polymerase chain reaction in conjunction with immunofluorescence were used to assess the expression and localization of Cx43 in hearts and cultured cardiomyocytes from wild-type and Gja1(Jrt/+) mice. Dye-coupling and dual whole cell patch-clamp recordings were also used to assess the gap junction channel status in cultured cardiomyocytes from wild-type and mutant mice. Cardiac tissue from adult Gja1(Jrt/+) mice revealed a 60-80% reduction in Cx43 protein with a preferential loss of the highly phosphorylated forms of Cx43. Compensation via the up-regulation of Cx40 or Cx45 was not observed. Immunofluorescent analysis of cultured cardiomyocytes revealed a trafficking defect, with a decrease in Cx43 plaques and a large population of Cx43 being retained in the Golgi apparatus. However, cultured cardiomyocytes from mutant mice remained beating with a 50% decrease in coupling conductance. CONCLUSION: These results suggest that the Cx43(G60S) mutant impairs normal trafficking and function of co-expressed Cx43 with no dramatic effect on cardiomyocyte function, suggesting that Cx43 is biosynthesized in excess of an essential need.

Decreased junctional adhesion molecule-A expression during blood-brain barrier breakdown.

Tight junctions between brain endothelial cells are one of the specialized structural components of the blood-brain barrier (BBB) to proteins. Research in the last decade has demonstrated that the integral membrane proteins of cerebral endothelial tight junctions are claudin, occludin, and junctional adhesion molecule (JAM). Altered expression of these tight junction proteins could cause BBB breakdown following brain injury leading to edema. In this study, expression of JAM-A, was analyzed by immunostaining and immunoblotting in the rat cortical cold injury model, a well-characterized in vivo model of BBB breakdown. Temporal and spatial expression of JAM-A was examined at 12 hours, days 2, 4, and 6 post-lesion in cold-injured and control rats. Control rats showed punctate JAM-A immunoreactivity at intervals along the circumference of the endothelial layer at tight junctions where JAM-A colocalized with occludin. A significant decrease in JAM-A expression was noted at the lesion site by immunoblotting at 12 h only. At this time period, lesion vessels showed loss of endothelial JAM-A immunostaining while day 2 onwards, there was recovery of endothelial JAM-A immunoreactivity. Dual labelling for JAM-A and fibronectin showed that only lesion vessels with BBB breakdown to fibronectin at 12 h also showed lack of endothelial JAM-A immunoreactivity supporting the evidence that JAM-A contributes to tight junction integrity.

Connexin levels regulate keratinocyte differentiation in the epidermis.

To understand the role of connexin43 (Cx43) in epidermal differentiation, we reduced Cx43 levels by RNA-mediated interference knockdown and impaired its functional status by overexpressing loss-of-function Cx43 mutants associated with the human disease oculodentodigital dysplasia (ODDD) in rat epidermal keratinocytes. When Cx43 expression was knocked down by 50-75%, there was a coordinate 55-65% reduction in Cx26 level, gap junction-based dye coupling was reduced by 60%, and transepithelial resistance decreased. Importantly, the overall growth and differentiation of Cx43 knockdown organotypic epidermis was severely impaired as revealed by alterations in the levels of the differentiation markers loricrin and involucrin and by reductions in vital and cornified layer thicknesses. Conversely, although the expression of Cx43 mutants reduced the coupling status of rat epidermal keratinocytes by approximately 80% without altering the levels of endogenous Cx43 or Cx26, their ability to differentiate was not altered. In addition, we used a mouse model of ODDD and found that newborn mice harboring the loss-of-function Cx43(G60S) mutant had slightly reduced Cx43 levels, whereas Cx26 levels, epidermis differentiation, and barrier function remained unaltered. This properly differentiated epidermis was maintained even when Cx43 and Cx26 levels decreased by more than 70% in 3-week-old mutant mice. Our studies indicate that Cx43 and Cx26 collectively co-regulate epidermal differentiation from basal keratinocytes but play a more minimal role in the maintenance of established epidermis. Altogether, these studies provide an explanation as to why the vast majority of ODDD patients, where Cx43 function is highly compromised, do not suffer from skin disease.

A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of doxorubicin against multidrug-resistant human breast cancer cells.

PURPOSE: This work is intended to develop and evaluate a new polymer-lipid hybrid nanoparticle system that can efficiently load and release water-soluble anticancer drug doxorubicin hydrochloride (Dox) and enhance Dox toxicity against multidrug-resistant (MDR) cancer cells. METHODS: Cationic Dox was complexed with a new soybean-oil-based anionic polymer and dispersed together with a lipid in water to form Dox-loaded solid lipid nanoparticles (Dox-SLNs). Drug loading and release properties were measured spectrophotometrically. The in vitro cytotoxicity of Dox-SLN and the excipients in an MDR human breast cancer cell line (MDA435/LCC6/MDR1) and its wild-type line were evaluated by trypan blue exclusion and clonogenic assays. Cellular uptake and retention of Dox were determined with a microplate fluorometer. RESULTS: Dox-SLNs were prepared with a drug encapsulation efficiency of 60-80% and a particle size range of 80-350 nm. About 50% of the loaded drug was released in the first few hours and an additional 10-20% in 2 weeks. Treatment of the MDR cells with Dox-SLN resulted in over 8-fold increase in cell kill when compared to Dox solution treatment at equivalent doses. The blank SLN and the excipients exhibited little cytotoxicity. The biological activity of the released Dox remained unchanged from fresh, free Dox. Cellular Dox uptake and retention by the MDR cells were both significantly enhanced (p < 0.05) when Dox was delivered in Dox-SLN form. CONCLUSIONS: The new polymer-lipid hybrid nanoparticle system is effective for delivery of Dox and enhances its efficacy against MDR breast cancer cells.

Functional characterization of oculodentodigital dysplasia-associated Cx43 mutants.

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.