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Thrombospondins (TSPs) are multidomain, secreted proteins that associate with cell surfaces and extracellular matrix. In mammals, there is a large body of data on functional roles of various TSP family members in cardiovascular disease (CVD), including stroke, cardiac remodeling and fibrosis, atherosclerosis, and aortic aneurysms. Coding single nucleotide polymorphisms (SNPs) of TSP1 or TSP4 are also associated with increased risk of several forms of CVD. Whereas interactions and functional effects of TSPs on a variety of cell types have been studied extensively, the molecular and cellular basis for the differential effects of the SNPs remains under investigation. Here, we provide an integrative review on TSPs, their roles in CVD and cardiovascular cell physiology, and known properties and mechanisms of TSP SNPs relevant to CVD. In considering recent expansions to knowledge of the fundamental cellular roles and mechanisms of TSPs, as well as the effects of wild-type and variant TSPs on cells of the cardiovascular system, we aim to highlight knowledge gaps and areas for future research or of translational potential.

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus (SON) whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganisation of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and become hyperphosphorylated following stimulation. By multi-omic integration of transcriptome and proteomic data we identify changes to proteins present in afferent inputs to this nucleus.Graphical abstract

The rising incidence of non-ST-segment elevation myocardial infarction (NSTEMI) and associated long-term high mortality constitutes an urgent clinical issue. Unfortunately, the study of possible interventions to treat this pathology lacks a reproducible pre-clinical model. Indeed, currently adopted small and large animal models of MI mimic only full-thickness, ST-segment-elevation (STEMI) infarcts, and hence cater only for investigation into therapeutics and interventions directed at this subset of MI. Thus, we developed an ovine model of NSTEMI by ligating the myocardial muscle at precise intervals parallel to the left anterior descending coronary artery. After validating the presented model both by histology and functional analysis with clinical data, further omics analyses highlighted the distinctive features of post-NSTEMI tissue remodelling. Here, by looking at the transcriptome and proteome-derived pathways emerging at acute (7 days) and late (28 days) post-surgery timepoints, we discovered specific alterations in cardiac post-ischaemic extracellular matrix (ECM). Together with the rise of well-known markers of inflammation and fibrosis, NSTEMI ischaemic regions showed distinctive patterns in the expression of complex N-glycans and glycosaminoglycans in cellular membranes and ECM. Identifying such changes in molecular moieties accessible to infusible and intra-myocardial injectable drugs sheds light on the development of targeted pharmacological solutions to contrast adverse fibrotic remodelling.

Background: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. Methods: Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. Results: We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. Conclusions: We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.

Ultradian glucocorticoid rhythms are highly conserved across mammalian species, however, their functional significance is not yet fully understood. Here we demonstrate that pulsatile corticosterone replacement in adrenalectomised rats induces a dynamic pattern of glucocorticoid receptor (GR) binding at ~3,000 genomic sites in liver at the pulse peak, subsequently not found during the pulse nadir. In contrast, constant corticosterone replacement induced prolonged binding at the majority of these sites. Additionally, each pattern further induced markedly different transcriptional responses. During pulsatile treatment, intragenic occupancy by active RNA polymerase II exhibited pulsatile dynamics with transient changes in enrichment, either decreased or increased depending on the gene, which mostly returned to baseline during the inter-pulse interval. In contrast, constant corticosterone exposure induced prolonged effects on RNA polymerase II occupancy at the majority of gene targets, thus acting as a sustained regulatory signal for both transactivation and repression of glucocorticoid target genes. The nett effect of these differences were consequently seen in the liver transcriptome as RNA-seq analysis indicated that despite the same overall amount of corticosterone infused, twice the number of transcripts were regulated by constant corticosterone infusion, when compared to pulsatile. Target genes that were found to be differentially regulated in a pattern-dependent manner were enriched in functional pathways including carbohydrate, cholesterol, glucose and fat metabolism as well as inflammation, suggesting a functional role for dysregulated glucocorticoid rhythms in the development of metabolic dysfunction., Author summary Adrenal glucocorticoid hormones are released in a characteristic ultradian rhythm that becomes dysregulated during chronic stress, disease, or synthetic corticosteroid treatment. Metabolic dysfunction is a comorbidity associated with all these conditions, but the role that altered glucocorticoid dynamics play is unknown. As the liver is a major site of glucocorticoid action on metabolic homeostasis regulated by the glucocorticoid receptor, we have assessed how different patterns of hormone replacement in adrenalectomised rats differentially regulate gene pathways involved in type II diabetes, cirrhosis, and fatty liver development, via altering the pattern of glucocorticoid receptor binding to regulatory sites. We believe our findings have important implications for therapies that can reproduce the endogenous glucocorticoid rhythm and thus minimize adverse metabolic side-effects in patients.

Diverticular disease (DD) is one of the most prevalent diseases of the large bowel. Lately, imbalance of neuro-muscular transmission has been recognized as a major etiological factor for DD. Neuronal calcitonin gene-related peptide (CGRP) is a potent gastrointestinal smooth muscle relaxant shown to have a widespread effect within the alimentary tract. Nevertheless, CGRPergic innervation of the enteric ganglia has never been considered in the context of motility impairment observed in DD patients. Changes in CGRP and calcitonin receptor-like receptor (CRLR) abundance within enteric ganglia were investigated in sigmoid samples from symptomatic and asymptomatic DD patients using quantitative fluorescence microscopy. CGRP effect on gastrointestinal smooth muscle was investigated using organ bath technique. We found CGRP levels within the enteric ganglia to be declined by up to 52% in symptomatic DD patients. Conversely, CRLR within the enteric ganglia was upregulated by 41% in symptomatic DD. Longitudinal smooth muscle displayed an elevated (+10.5%) relaxant effect to the exogenous application of CGRP in colonic strips from symptomatic DD patients. Samples from asymptomatic DD patients consistently showed intermediate values across different experiments. In conclusion, the present study demonstrates that CGRPergic signaling is subject to alteration in DD. Our results suggest that a hypersensitization mechanism to gradually decreasing levels of CGRP-IR nerve fibers takes place during DD progression. Alterations to CGRPergic signaling in DD disease may have implications for physiological abnormalities associated with colonic DD.

Although the pig is a model for heart disease, the neuroanatomy of cardiac ventricles (CV) in this species remains undetailed. We aimed to define the innervation pattern of pig CV, combining histochemistry for acetylcholinesterase, immunofluorescent labeling and electron microscopy. Forty nine examined pig hearts show that the major nerves supplying the ventral side of CV descend from the venous part of the heart hilum. Fewer in number and smaller in size, epicardial nerves supply the dorsal half of the CV. Epicardial nerves on the left ventricle are thicker than those on the right. Ventricular ganglia of various sizes distribute at the basal level of both CV. Averagely, we found 3,848 ventricular neuronal somata per heart. The majority of somata were cholinergic, although ganglionic cells of different neurochemical phenotypes (positive for nNOS, ChAT/nNOS, or ChAT/TH) were also observed. Large and most numerous nerves proceeded within the epicardium. Most of endocardium and myocardium contained a network of nerve bundles and nerve fibers (NFs). But, a large number of thin nerves extended along the bundle of His and its branches. The majority of NFs were adrenergic, while cholinergic NFs were scarce yet more abundant than nitrergic ones. Sensory NFs positive for CGRP were the second most abundant phenotype after adrenergic NFs in all layers of the ventricular wall. Electron microscopy elucidated that ultrastructure of nerves varied between different areas of CV. The described structural organization of CV provides an anatomical basis for further functional and pathophysiological studies in the pig heart. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1756-1780, 2017. © 2017 Wiley Periodicals, Inc.

Background Cardiac autonomic neuropathy is thought to cause adverse cardiovascular effects in diabetes mellitus. Pulmonary vein ganglia ( PVG ), which have been implicated in normal and abnormal heart rhythm regulation, have not been fully investigated in type 1 diabetes mellitus (T1D). We examined the functional and anatomical effects of T1D on PVG and studied the details of T1D‐induced remodeling on the PVG structure and function. Methods and Results We used a mouse model of T1D (Akita mouse), immunofluorescence, isolated Langendorff‐perfused hearts, and mathematical simulations to explore the effects of T1D on PVG . Whole‐mount atrial immunofluorescence of choline acetyltransferase and tyrosine hydroxylase labeling showed that sympathetic and parasympathetic somas of the PVG neurons were significantly hypotrophied in T1D hearts versus wild type. Stimulation of PVG in isolated Langendorff‐perfused hearts caused more pronounced P‐P interval prolongation in wild type compared with Akita hearts. Propranolol resulted in a comparable P‐P prolongation in both phenotypes, and atropine led to more pronounced P‐P interval shortening in wild type compared with Akita hearts. Numerical modeling using network simulations revealed that a decrease in the sympathetic and parasympathetic activities of PVG in T1D could explain the experimental results. Conclusions T1D leads to PVG remodeling with hypotrophy of sympathetic and parasympathetic cell bodies and a concomitant decrease in the PVG sympathetic and parasympathetic activities.

Fluorescent immunohistochemistry on the cardiac conduction system in whole mount mouse heart preparations demonstrates a particularly dense and complex network of nerve fibres and cardiomyocytes which are positive to the hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4-positive cardiomyocytes) in the sinoatrial node region and adjacent areas around the root of right cranial vein. The present study was designed to investigate the morphologic and histochemical pattern of nerve fibres and HCN4-positive cardiomyocytes using fluorescent techniques and/or electron microscopy. Adrenergic and cholinergic nerve fibres together with HCN4-positive cardiomyocytes were identified using primary antibodies for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and the HCN4 channel respectively. Amid HCN4-positive cardiomyocytes, fluorescence and electron microscopy data demonstrated a dense distribution of nerve fibres immunoreactive for ChAT and TH. In addition, novel electron microscopy data revealed that the mouse sinoatrial node contained exclusively unmyelinated nerve fibres, in which the majority of axons possess varicosities with clear mediatory vesicles that can be classified as cholinergic. Synapses occurred without any clear terminal connection with the effector cell, i.e. these synapes were of "en passant" type. In general, the morphologic pattern of innervation of mouse HCN4-positive cardiomyocytes identified using electron microscopy corresponds well to the dense network of nerve fibres demonstrated by fluorescent immunohistochemistry in mouse sinoatrial node and adjacent areas. The complex and extraordinarily dense innervation of HCN4-positive cardiomyocytes in mouse sinoatrial node underpins the importance of neural regulation for the cardiac conduction system. Based on the present observations, it is concluded that the occurrence of numerous nerve fibres nearby atrial cardiomyocytes serves as a novel reliable extracellular criterion for discrimination of SA nodal cardiomyocytes using electron microscopy.

Although the rabbit is routinely used as the animal model of choice to investigate cardiac electrophysiology, the neuroanatomy of the rabbit heart is not well documented. The aim of this study was to examine the topography of the intrinsic nerve plexus located on the rabbit heart surface and interatrial septum stained histochemically for acetylcholinesterase using pressure-distended whole hearts and whole-mount preparations from 33 Californian rabbits. Mediastinal cardiac nerves entered the venous part of the heart along the root of the right cranial vein (superior caval vein) and at the bifurcation of the pulmonary trunk. The accessing nerves of the venous part of the heart passed into the nerve plexus of heart hilum at the heart base. Nerves approaching the heart extended epicardially and innervated the atria, interatrial septum and ventricles by five nerve subplexuses, i.e. left and middle dorsal, dorsal right atrial, ventral right and left atrial subplexuses. Numerous nerves accessed the arterial part of the arterial part of the heart hilum between the aorta and pulmonary trunk, and distributed onto ventricles by the left and right coronary subplexuses. Clusters of intrinsic cardiac neurons were concentrated at the heart base at the roots of pulmonary veins with some positioned on the infundibulum. The mean number of intrinsic neurons in the rabbit heart is not significantly affected by aging: 2200 ± 262 (range 1517–2788; aged) vs. 2118 ± 108 (range 1513–2822; juvenile). In conclusion, despite anatomic differences in the distribution of intrinsic cardiac neurons and the presence of well-developed nerve plexus within the heart hilum, the topography of all seven subplexuses of the intrinsic nerve plexus in rabbit heart corresponds rather well to other mammalian species, including humans.

Although the pig is a model for heart disease, the neuroanatomy of cardiac ventricles (CV) in this species remains undetailed. We aimed to define the innervation pattern of pig CV, combining histochemistry for acetylcholinesterase, immunofluorescent labeling and electron microscopy. Forty nine examined pig hearts show that the major nerves supplying the ventral side of CV descend from the venous part of the heart hilum. Fewer in number and smaller in size, epicardial nerves supply the dorsal half of the CV. Epicardial nerves on the left ventricle are thicker than those on the right. Ventricular ganglia of various sizes distribute at the basal level of both CV. Averagely, we found 3,848 ventricular neuronal somata per heart. The majority of somata were cholinergic, although ganglionic cells of different neurochemical phenotypes (positive for nNOS, ChAT/nNOS, or ChAT/TH) were also observed. Large and most numerous nerves proceeded within the epicardium. Most of endocardium and myocardium contained a network of nerve bundles and nerve fibers (NFs). But, a large number of thin nerves extended along the bundle of His and its branches. The majority of NFs were adrenergic, while cholinergic NFs were scarce yet more abundant than nitrergic ones. Sensory NFs positive for CGRP were the second most abundant phenotype after adrenergic NFs in all layers of the ventricular wall. Electron microscopy elucidated that ultrastructure of nerves varied between different areas of CV. The described structural organization of CV provides an anatomical basis for further functional and pathophysiological studies in the pig heart. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1756-1780, 2017. © 2017 Wiley Periodicals, Inc.

The rabbit is widely used in experimental cardiac physiology, but the neuroanatomy of the rabbit heart remains insufficiently examined. This study aimed to ascertain the architecture of the intrinsic nerve plexus in the walls and septum of rabbit cardiac ventricles. In 51 rabbit hearts, a combined approach involving: (i) histochemical acetylcholinesterase staining of intrinsic neural structures in total cardiac ventricles; (ii) immunofluorescent labelling of intrinsic nerves, nerve fibres (NFs) and neuronal somata (NS); and (iii) transmission electron microscopy of intrinsic ventricular nerves and NFs was used. Mediastinal nerves access the ventral and lateral surfaces of both ventricles at a restricted site between the root of the ascending aorta and the pulmonary trunk. The dorsal surface of both ventricles is supplied by several epicardial nerves extending from the left dorsal ganglionated nerve subplexus on the dorsal left atrium. Ventral accessing nerves are thicker and more numerous than dorsal nerves. Intrinsic ventricular NS are rare on the conus arteriosus and the root of the pulmonary trunk. The number of ventricular NS ranged from 11 to 220 per heart. Four chemical phenotypes of NS within ventricular ganglia were identified, i.e. ganglionic cells positive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and biphenotypic, i.e. positive for both ChAT/nNOS and for ChAT/tyrosine hydroxylase. Clusters of small intensely fluorescent cells are distributed within or close to ganglia on the root of the pulmonary trunk, but not on the conus arteriosus. The largest and most numerous intrinsic nerves proceed within the epicardium. Scarce nerves were found near myocardial blood vessels, but the myocardium contained only a scarce meshwork of NFs. In the endocardium, large numbers of thin nerves and NFs proceed along the bundle of His and both its branches up to the apex of the ventricles. The endocardial meshwork of fine NFs was approximately eight times denser than the myocardial meshwork. Adrenergic NFs predominate considerably in all layers of the ventricular walls and septum, whereas NFs of other neurochemical phenotypes were in the minority and their amount differed between the epicardium, myocardium and endocardium. The densities of NFs positive for nNOS and ChAT were similar in the epicardium and endocardium, but NFs positive for nNOS in the myocardium were eight times more abundant than NFs positive for ChAT. Potentially sensory NFs positive for both calcitonin gene-related peptide and substance P were sparse in the myocardial layer, but numerous in epicardial nerves and particularly abundant within the endocardium. Electron microscopic observations demonstrate that intrinsic ventricular nerves have a distinctive morphology, which may be attributed to remodelling of the peripheral nerves after their access into the ventricular wall. In conclusion, the rabbit ventricles display complex structural organization of intrinsic ventricular nerves, NFs and ganglionic cells. The results provide a basic anatomical background for further functional analysis of the intrinsic nervous system in the cardiac ventricles.

Neuroanatomy of cardiac ventricles in bats that are adapted for 5-7 month long hibernation was not identified so far. We used fluorescent immunolabeling for PGP9.5, TH, ChAT, nNOS, SP, and CGRP in ...

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

In spite of numerous advances in understanding diverticular disease, its pathogenesis remains one of the main problems to be solved. We aimed to investigate the ultrastructural changes of the enteric nervous system in unaffected individuals, in asymptomatic patients with diverticulosis and in patients with diverticular disease.Transmission electron microscopy was used to analyse samples of the myenteric, outer submucosal and inner submucosal plexuses from patients without diverticula (n=9), asymptomatic patients with diverticulosis (n=7) and in patients with complicated diverticular disease (n=9). We described the structure of ganglia, interstitial cells of Cajal and enteric nerves, as well as their relationship with each other. The distribution and size of nerve processes were analysed quantitatively.In complicated diverticular disease, neurons exhibited larger lipofuscin-like inclusions, their membranous organelles had larger cisterns and the nucleus showed deeper indentations. Nerve remodeling occurred in every plexus, characterised by an increased percentage of swollen and fine neurites. Interstitial cells of Cajal had looser contacts with the surrounding cells and showed cytoplasmic depletion and proliferation of the rough endoplasmic reticulum. In asymptomatic patients with diverticulosis, alterations of enteric nerves and ICC were less pronounced.In conclusion, the present findings suggest that most ultrastructural changes of the enteric nervous system occur in complicated diverticular disease. The changes are compatible with damage to the enteric nervous system and reactive remodeling of enteric ganglia, nerves and interstitial cells of Cajal. Disrupted architecture of enteric plexuses might explain clinical and pathophysiological changes associated with diverticular disease.