Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.

The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block-face scanning electron microscopy (SBF-SEM), and computational image reconstruction, to enable the first three-dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF-SEM and up to 150 serial scanning electron microscopy images (covering z-axes distances of 12-8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath-like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte-endothelial interactions with direct contact points and peg-and-socket-like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software-generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell-to-cell closeness, has enabled preliminary semi-quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte-endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing.

Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αßγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δßγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

Regional identification of information flow termination of electrohysterographic signals: Towards understanding human uterine electrical propagation.

BACKGROUND AND OBJECTIVE: The uterine electrohysterogram (EHG) contains important information about electrical signal propagation which may be useful to monitor and predict the progress of pregnancy towards parturition. Directed information processing has the potential to be of use in studying EHG recordings. However, so far, there is no directed information-based estimation scheme that has been applied to investigating the propagation of human EHG recordings. To realize this, the approach of directed information and its reliability and adaptability should be scientifically studied. METHODS: We demonstrated an estimation scheme of directed information to identify the spatiotemporal relationship between the recording channels of EHG signal and assess the algorithm reliability initially using simulated data. Further, a regional identification of information flow termination (RIIFT) approach was developed and applied for the first time to extant multichannel EHG signals to reveal the terminal zone of propagation of the electrical activity associated with uterine contraction. RIIFT operates by estimating the pairwise directed information between neighboring EHG channels and identifying the location where there is the strongest inward flow of information. The method was then applied to publicly-available experimental data obtained from pregnant women with the use of electrodes arranged in a 4-by-4 grid. RESULTS: Our results are consistent with the suggestions from the previous studies with the added identification of preferential sites of excitation termination - within the estimated area, the direction of surface action potential propagation towards the medial axis of uterus during contraction was discovered for 72.15% of the total cases, demonstrating that our RIIFT method is a potential tool to investigate EHG propagation for advancing our understanding human uterine excitability. CONCLUSIONS: We developed a new approach and applied it to multichannel human EHG recordings to investigate the electrical signal propagation involved in uterine contraction. This provides an important platform for future studies to fill knowledge gaps in the spatiotemporal patterns of electrical excitation of the human uterus.

Strategies for Peptide-Mediated Cargo Delivery to Human Smooth Muscle Cells.

Diseases involving dysfunction of smooth muscle cells present a major health and socioeconomic burden, and have remained stubbornly resistant to standard therapeutic strategies. Examples include many cardiovascular diseases and spontaneous preterm birth, a complication affecting up to 11% of all pregnancies worldwide. This fuels the continued search for new drug delivery strategies to treat these conditions. The use of cell penetrating peptides (CPPs) for this purpose remains a promising, if as yet unrealized, avenue to explore. In part, this may relate to a paucity of studies investigating the application of CPPs as drug delivery vectors to human smooth muscle cells and tissues. We have sought to address this knowledge gap by reporting methods for examining the uptake of different CPP-cargo vectors to human uterine and vascular smooth muscle cells. In particular, we report here (a) that four different CPP-fluorophore conjugates, spanning masses of 1309-3435 Da, and net charges of +2 to +7, can be delivered to human isolated uterine smooth muscle cells without inducing cell toxicity; (b) that the cargo delivered by such CPPs can be fluorescent moieties and/or biologically active peptides; (c) that CPP delivery in a short time frame to native smooth muscle cells in human tissues ex vivo can be achieved. Further exploration of CPPs as tools to facilitate targeted drug delivery to native human smooth muscle tissues will assist in improving our understanding of scientific mechanisms underlying major diseases involving smooth muscle dysfunction as well as facilitating therapeutic investigations.

The Generation of a Comprehensive Spectral Library for the Analysis of the Guinea Pig Proteome by SWATH-MS.

Advances in liquid chromatography-mass spectrometry have facilitated the incorporation of proteomic studies to many biology experimental workflows. Data-independent acquisition platforms, such as sequential window acquisition of all theoretical mass spectra (SWATH-MS), offer several advantages for label-free quantitative assessment of complex proteomes over data-dependent acquisition (DDA) approaches. However, SWATH data interpretation requires spectral libraries as a detailed reference resource. The guinea pig (Cavia porcellus) is an excellent experimental model for translation to many aspects of human physiology and disease, yet there is limited experimental information regarding its proteome. To overcome this knowledge gap, a comprehensive spectral library of the guinea pig proteome is generated. Homogenates and tryptic digests are prepared from 16 tissues and subjected to >200 DDA runs. Analysis of >250 000 peptide-spectrum matches resulted in a library of 73 594 peptides from 7666 proteins. Library validation is provided by i) analyzing externally derived SWATH files (https://doi.org/10.1016/j.jprot.2018.03.023) and comparing peptide intensity quantifications; ii) merging of externally derived data to the base library. This furnishes the research community with a comprehensive proteomic resource that will facilitate future molecular-phenotypic studies using (re-engaging) the guinea pig as an experimental model of relevance to human biology. The spectral library and raw data are freely accessible in the MassIVE repository (MSV000083199).

Inhibition of Inflammatory Changes in Human Myometrial Cells by Cell Penetrating Peptide and Small Molecule Inhibitors of NFκB.

Complications arising from Preterm Birth are the leading causes of neonatal death globally. Current therapeutic strategies to prevent Preterm Birth are yet to demonstrate success in terms of reducing this neonatal disease burden. Upregulation of intracellular inflammatory pathways in uterine cells, including those involving nuclear factor kappa-B (NFκB), have been causally linked to both human term and preterm labor, but the barrier presented by the cell membrane presents an obstacle to interventions aimed at dampening these inflammatory responses. Cell penetrating peptides (CPPs) are novel vectors that can traverse cell membranes without the need for recognition by cell surface receptors and offer the ability to deliver therapeutic cargo internal to cell membranes. Using a human uterine cell culture inflammatory model, this study aimed to test the effectiveness of CPP-cargo delivery to inhibit inflammatory responses, comparing this effect with a small molecule inhibitor (Sc514) that has a similar intracellular target of action within the NFκB pathway (the IKK complex). The CPP Penetratin, conjugated to rhodamine, was able to enter uterine cells within a 60 min timeframe as assessed by live confocal microscopy, this phenomena was not observed with the use of a rhodamine-conjugated inert control peptide (GC(GS)4). Penetratin CPP conjugated to an IKK-inhibitory peptide (Pen-NBD) demonstrated ability to inhibit both the IL1ß-induced expression of the inflammatory protein COX2 and dampen the expression of a bespoke array of inflammatory genes. Truncation of the CPP vector rendered the CPP-cargo conjugate much less effective, demonstrating the importance of careful vector selection. The small molecule inhibitor Sc514 also demonstrated ability to inhibit COX2 protein responses and a broad down-regulatory effect on uterine cell inflammatory gene expression. These results support the further exploration of either CPP-based or small molecular treatment strategies to dampen gestational cell inflammatory responses in the context of preterm birth. The work underlines both the importance of careful selection of CPP vector-cargo combinations and basic testing over a broad time and concentration range to ensure effective responses. Further work should demonstrate the effectiveness of CPP-linked cargos to dampen alternative pathways of inflammation linked to Preterm Birth such as MAP Kinase or AP1.

Depletion of Myofibril-Associated Proteins Using Selective Protein Extraction as a Tool in Cardiac Proteomics.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a long-established EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.

Estrogenic vascular effects are diminished by chronological aging.

The beneficial role of estrogen in the vascular system may be due, in part, through reduction of peripheral vascular resistance. The use of estrogen therapy to prevent cardiovascular disease in post-menopausal women remains contentious. This study investigated the influence of aging and the menopause on the acute vasodilatory effects of estrogen using ex vivo human and murine resistance arteries. Vessels were obtained from young (2.9 ± 0.1 months) and aged (24.2 ± 0.1 and 28.9 ± 0.3 months) female mice and pre- (42.3 ± 0.5 years) and post-menopausal (61.9 ± 0.9 years) women. Aging was associated with profound structural alterations of murine uterine arteries, including the occurrence of outward hypertrophic remodeling and increased stiffness. Endothelial and smooth muscle function were diminished in uterine (and tail) arteries from aged mice and post-menopausal women. The acute vasodilatory effects of 17ß-estradiol (non-specific estrogen receptor (ER) agonist), PPT (ERα-specific agonist) and DPN (ERß-specific agonist) on resistance arteries were attenuated by aging and the menopause. However, the impairment of estrogenic relaxation was evident after the occurrence of age-related endothelial dysfunction and diminished distensibility. The data indicate, therefore, that chronological resistance arterial aging is a prominent factor leading to weakened vasodilatory action of estrogenic compounds.

Nuclear factor of activated T-cell isoform expression and regulation in human myometrium.

BACKGROUND: During pregnancy, myometrial gene and protein expression is tightly regulated to accommodate fetal growth, promote quiescence and ultimately prepare for the onset of labour. It is proposed that changes in calcium signalling, may contribute to regulating gene expression and that nuclear factor of activated T-cell (NFAT) transcription factors (isoforms c1-c4) may be involved. Currently, there is little information regarding NFAT expression and regulation in myometrium. METHODS: This study examined NFAT isoform mRNA expression in human myometrial tissue and cells from pregnant women using quantitative PCR. The effects of the Ca(2+) ionophore A23187 and in vitro stretch (25 % elongation, static strain; Flexercell FX-4000 Tension System) on NFAT expression were determined in cultured human myometrial cells. RESULTS: Human myometrial tissue and cultured cells expressed NFATc1-c4 mRNA. NFATc2 gene expression in cultured cells was increased in response to 6 h stretch (11.5 fold, P < 0.001, n = 6) and calcium ionophore (A23187, 5 µM) treatment (20.6 fold, P < 0.001, n = 6). This response to stretch was significantly reduced (90 %, P < 0.001, n = 10) in the presence of an intracellular calcium chelator, BAPTA-AM (20 µM). CONCLUSIONS: These data suggest that NFATc2 expression is regulated by intracellular calcium and in vitro stretch, and that the stretch response in human myometrial cells is dependent upon intracellular calcium signalling pathways. Our findings indicate a potentially unique role for NFATc2 in mediating stretch-induced gene expression per se and warrant further exploration in relation to the mechanisms promoting uterine smooth muscle growth in early pregnancy and/or labour.

Chemotactic activity of gestational tissues through late pregnancy, term labor, and RU486-induced preterm labor in Guinea pigs.

PROBLEM: Is increased leukocyte chemotactic activity (CA) from gestational tissues necessary for term or preterm labor in guinea pigs? METHOD OF STUDY: Tissue extracts were prepared from pregnant guinea pig decidua-myometrium, cervix, fetal membranes (amniochorion), and placenta during early third trimester (n = 8), term not in labor (TNL, n = 5), and term spontaneous labor (TL, n = 6), RU486-induced preterm labor (PTL, n = 6), or controls (cPTL, n = 5). Leukocyte CA was assessed using a modified Boyden chamber assay. Extract chemokine and maternal progesterone concentrations were quantified by enzyme immunoassay. RESULTS: Only the extracts from amniochorion demonstrated increased CA through late gestation and labor. In contrast, CA was decreased in extracts from amniochorion and cervix from animals after RU486-induced PTL. Maternal progesterone concentrations remained high in all groups. CONCLUSION: Leukocyte CA of intrauterine tissues is increased in term spontaneous labor. However, RU486-induced preterm labor occurs in the absence of increased CA.

Computational modeling reveals key contributions of KCNQ and hERG currents to the malleability of uterine action potentials underpinning labor.

The electrical excitability of uterine smooth muscle cells is a key determinant of the contraction of the organ during labor and is manifested by spontaneous, periodic action potentials (APs). Near the end of term, APs vary in shape and size reflecting an ability to change the frequency, duration and amplitude of uterine contractions. A recent mathematical model quantified several ionic features of the electrical excitability in uterine smooth muscle cells. It replicated many of the experimentally recorded uterine AP configurations but its limitations were evident when trying to simulate the long-duration bursting APs characteristic of labor. A computational parameter search suggested that delayed rectifying K(+) currents could be a key model component requiring improvement to produce the longer-lasting bursting APs. Of the delayed rectifying K(+) currents family it is of interest that KCNQ and hERG channels have been reported to be gestationally regulated in the uterus. These currents exhibit features similar to the broadly defined uterine IK1 of the original mathematical model. We thus formulated new quantitative descriptions for several I(KCNQ) and I(hERG). Incorporation of these currents into the uterine cell model enabled simulations of the long-lasting bursting APs. Moreover, we used this modified model to simulate the effects of different contributions of I(KCNQ) and I(hERG) on AP form. Our findings suggest that the alterations in expression of hERG and KCNQ channels can potentially provide a mechanism for fine tuning of AP forms that lends a malleability for changing between plateau-like and long-lasting bursting-type APs as uterine cells prepare for parturition.

Computational modeling of inhibition of voltage-gated Ca channels: identification of different effects on uterine and cardiac action potentials.

The uterus and heart share the important physiological feature whereby contractile activation of the muscle tissue is regulated by the generation of periodic, spontaneous electrical action potentials (APs). Preterm birth arising from premature uterine contractions is a major complication of pregnancy and there remains a need to pursue avenues of research that facilitate the use of drugs, tocolytics, to limit these inappropriate contractions without deleterious actions on cardiac electrical excitation. A novel approach is to make use of mathematical models of uterine and cardiac APs, which incorporate many ionic currents contributing to the AP forms, and test the cell-specific responses to interventions. We have used three such models-of uterine smooth muscle cells (USMC), cardiac sinoatrial node cells (SAN), and ventricular cells-to investigate the relative effects of reducing two important voltage-gated Ca currents-the L-type (ICaL) and T-type (ICaT) Ca currents. Reduction of ICaL (10%) alone, or ICaT (40%) alone, blunted USMC APs with little effect on ventricular APs and only mild effects on SAN activity. Larger reductions in either current further attenuated the USMC APs but with also greater effects on SAN APs. Encouragingly, a combination of ICaL and ICaT reduction did blunt USMC APs as intended with little detriment to APs of either cardiac cell type. Subsequent overlapping maps of ICaL and ICaT inhibition profiles from each model revealed a range of combined reductions of ICaL and ICaT over which an appreciable diminution of USMC APs could be achieved with no deleterious action on cardiac SAN or ventricular APs. This novel approach illustrates the potential for computational biology to inform us of possible uterine and cardiac cell-specific mechanisms. Incorporating such computational approaches in future studies directed at designing new, or repurposing existing, tocolytics will be beneficial for establishing a desired uterine specificity of action.

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility.

Recurring patterns in stationary intervals of abdominal uterine electromyograms during gestation.

Abdominal uterine electromyograms (uEMG) studies have focused on uterine contractions to describe the evolution of uterine activity and preterm birth (PTB) prediction. Stationary, non-contracting uEMG has not been studied. The aim of the study was to investigate the recurring patterns in stationary uEMG, their relationship with gestation age and PTB, and PTB predictivity. A public database of 300 (38 PTB) three-channel (S1-S3) uEMG recordings of 30 min, collected between 22 and 35 weeks' gestation, was used. Motion and labour contraction-free intervals in uEMG were identified as 5-min weak-sense stationarity intervals in 268 (34 PTB) recordings. Sample entropy (SampEn), percentage recurrence (PR), percentage determinism (PD), entropy (ER), and maximum length (L MAX) of recurrence were calculated and analysed according to the time to delivery and PTB. Random time series were generated by random shuffle (RS) of actual data. Recurrence was present in actual data (p<0.001) but not RS. In S3, PR (p<0.005), PD (p<0.01), ER (p<0.005), and L MAX (p<0.05) were higher, and SampEn lower (p<0.005) in PTB. Recurrence indices increased (all p<0.001) and SampEn decreased (p<0.01) with decreasing time to delivery, suggesting increasingly regular and recurring patterns with gestation progression. All indices predicted PTB with AUC≥0.62 (p<0.05). Recurring patterns in stationary non-contracting uEMG were associated with time to delivery but were relatively poor predictors of PTB.

Differentiation of human epidermal neural crest stem cells (hEPI-NCSC) into virtually homogenous populations of dopaminergic neurons.

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons, which degenerate in Parkinson's disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells, followed by ventralizing, patterning, continued exposure to the TGFß receptor inhibitor, SB431542, and at later stages of differentiation the presence of the WNT inhibitor, IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification, with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type, hEPI-NCSC.