Experimental and Computational Insight Into Human Mesenchymal Stem Cell Paracrine Signaling and Heterocellular Coupling Effects on Cardiac Contractility and Arrhythmogenicity.

RATIONALE: Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved. OBJECTIVE: The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches. METHODS AND RESULTS: Extending our previous hMSC-cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation-contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non-hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). CONCLUSIONS: Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility.

The paracrine control of vascular motion. A historical perspective.

During the last quarter of the past century, the leading role the endocrine and nervous systems had on the regulation of vasomotion, shifted towards a more paracrine-based regulation. This begun with the recognition of endothelial cells as active players of vascular control, when the vessel's intimal layer was identified as the main source of prostacyclin and was followed by the discovery of an endothelium-derived smooth muscle cell relaxing factor (EDRF). The new position acquired by endothelial cells prompted the discovery of other endothelium-derived regulatory products: vasoconstrictors, generally known as EDCFs, endothelin, and other vasodilators with hyperpolarizing properties (EDHFs). While this research was taking place, a quest for the discovery of the nature of EDRF carried back to a research line commenced a decade earlier: the recently found intracellular messenger cGMP and nitrovasodilators. Both were smooth muscle relaxants and appeared to interact in a hormonal fashion. Prejudice against an unconventional gaseous molecule delayed the acceptance that EDRF was nitric oxide (NO). When this happened, a new era of research that exceeded the vascular field commenced. The discovery of the pathway for NO synthesis from L-arginine involved the clever assembling of numerous unrelated observations of different areas of knowledge. The last ten years of research on the paracrine regulation of the vascular wall has shifted to perivascular fat (PVAT), which is beginning to be regarded as the fourth layer of the vascular wall. Starting with the discovery of an adipose-derived relaxing substance (ADRF), the role that different adipokines have on the paracrine control of vasomotion is now filling the research activity of many vascular pharmacology labs, and surprising interactions between the endothelium, PVAT and smooth muscle are being unveiled.

Vascular smooth muscle cell optimization of vasculogenesis within naturally derived, biodegradable, hybrid hydrogel scaffolds.

BACKGROUND: As vascularization represents the rate-limiting step in permanent incorporation of hydrogel-based tissue-regeneration templates, the authors sought to identify the material chemistry that would optimize endothelial cell adhesion and invasion into custom hydrogel constructs. The authors further investigated induction of endothelial tubule formation by growth factor supplementation and paracrine stimulation. METHODS: Hydrogel scaffolds consisting of combinations of alginate, collagen type I, and chitosan were seeded with human umbilical vein endothelial cells and maintained under standard conditions for 14 days. Cell density and invasion were then evaluated. Tubule formation was evaluated following basic fibroblast growth factor addition or co-culture with human aortic smooth muscle cells. RESULTS: Human umbilical vein endothelial cells demonstrated greatest cell-surface density and invasion volumes with alginate and collagen (10:1 weight/weight) scaffolds (p < 0.05). Supplementation with basic fibroblast growth factor increased surface density but neither invasion nor tubule formation. A significant increase in tubule content/organization was observed with increasing human aortic smooth muscle cell-to-human umbilical vein endothelial cell ratio co-culture. CONCLUSIONS: Alginate and collagen 10:1 scaffolds allow for maximal cellularization compared with other combinations studied. Growth factor supplementation did not affect human umbilical vein endothelial cell invasion or morphology. Paracrine signaling by means of co-culture with human umbilical vein endothelial cells stimulated endothelial tubule formation and vascular protonetwork organization. These findings serve to guide future endeavors toward fabrication of prevascularized tissue constructs.

Paracrine Hedgehog increases the steroidogenic potential of prostate stromal cells in a Gli-dependent manner.

Acquired intratumoral steroidogenesis is involved in progression of prostate cancer to castration resistant disease (CRPC) and a target for improved therapeutics. Recent work has shown that prostate cancer cells can acquire steroidogenic activity as they progress to a therapeutic-resistant state. However, benign prostate stromal cells (PrSCs) also have steroidogenic potential though they are often overlooked as a source of intratumoral androgens. Here, we present preliminary studies showing that the steroidogenic activity of primary human PrSCs is significantly increased by exposure to a Hedgehog agonist (SAG) or by transduction of PrSCs with lentiviruses that expresses active Gli2 (Gli2ΔN), a transcription factor that is triggered by Hh signaling. Comparative gene expression profiling on Chips, that was confirmed by quantitative real-time PCR, revealed that hedgehog agonist treatment induced in these cells expressions of hedgehog target genes (Gli1, Ptch1, and SCUBE1) plus a specific cadre of genes involved in cholesterol/steroid biosynthesis, metabolism, and transport. Genes involved downstream in steroid hormone generation, including CYP17A1 and CYP19A1 were also induced. Both the hedgehog agonist and the Gli2-expressing lentivirus significantly increased the output of testosterone (T) from PrSCs that were supplemented with dihydroepiandrosterone (DHEA), an adrenal precursor of T. Finally, knockdown of Gli2 by siRNA suppressed the ability of SAG to induce this response. Collectively, our data indicate that hedgehog/Gli signaling may be a factor in acquired intratumoral steroidogenesis of a prostate tumor through its actions on stromal cells in the tumor microenvironment and an influence for the development of CRPC.

Paracrine regulation of growth hormone secretion by estrogen in women.

CONTEXT: Paracrine regulation is emerging as a discrete control mechanism in the endocrine system. In hypogonadal men, stimulation of GH secretion by testosterone requires prior aromatization to estradiol, a paracrine effect unmasked by central estrogen receptor blockade with tamoxifen. In hypogonadal women, estrogen replacement via a physiological non-oral route fails to enhance GH secretion, indicating an absence of an endocrine effect. The aim was to investigate whether local estrogens produced from aromatization regulate GH secretion. DESIGN: We conducted an open-label, two-phase, crossover study. PATIENTS AND INTERVENTION: We compared the effects on GH secretion of tamoxifen with estradiol valerate in postmenopausal women. Ten women were treated with tamoxifen (10 and 20 mg/d) and estradiol valerate (2 mg/d) via oral route for 2 wk each, with a washout period of at least 6 wk. MAIN OUTCOME MEASURES: We measured the GH response to arginine and circulating levels of IGF-I and SHBG, markers of hepatic estrogen effect. RESULTS: The GH response to arginine was reduced by 10- and 20-mg tamoxifen in a dose-dependent manner and potentiated significantly (P<0.05) by estradiol valerate. Mean IGF-I concentration was reduced significantly with high-dose tamoxifen (P<0.01) and estradiol valerate treatment (P<0.05), whereas mean SHBG levels rose with both (P<0.01). CONCLUSIONS: Blunted GH response to stimulation occurring in the face of reduced IGF-I feedback inhibition with tamoxifen indicates that GH secretion was suppressed by estrogen receptor antagonism. Because circulating estradiol was unaffected, these data indicate a significant role of local estrogen in the central control of GH secretion. We conclude that aromatase mediates the paracrine control of GH secretion in women.

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

[Regulatory effect of Ningxin Hongqi Capsule on local ovarian autocrine and paracrine factors in rats during peri-menopausal period].

OBJECTIVE: To explore the regulatory effect and mechanism of Ningxin Hongqi Capsule on local ovarian autocrine and paracrine factors in peri-menopausal rats. METHODS: SD female rats aged 4 months were allocated in a normal control group (A) and those aged 14 months with vagino-cytologic figure of oestrus elongation were allocated in a senile female rat model group (B). Rats in Group B were subdivided into 5 groups randomly as the B1, B2 and B3 subgroups treated respectively with high, moderate and low dose Ningxin Hongqi Capsule, the B4 subgroup treated with estradiol and the B5 subgroup untreated for control. Rats' ovaries were obtained at the end of the experiment for observing the conditions of ovarian growing follicles and corpus luteum by HE staining, determining expressions of ovarian estradiol receptor (ER), progesterone receptor (PR), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin alpha (INHalpha), activin (ACT) alpha-beta, follistatin (FS), and insulin-like growth factor (IGF-1). RESULTS: As compared with Group B5, the ovary index, number of growing follicle were higher and levels of FSH and LH were lower in Group B2 and B3, expression of ER was higher in Group B1 and B4, IGF-1 and INHalpha was higher in Group B2 and B3, and ACTalpha-beta and FS were lower (all P < 0.05). CONCLUSION: Nirigxin Hongqi Capsule could adjust and balance the local ovarian autocrine and paracrine factors to improve the ovarian function.

Basic developmental rules and their implications for epilepsy in the immature brain.

The construction of the human brain with its 10(15) synapses follows a set of complex developmentally and environmentally regulated steps. A series of sequences have been described that are instrumental, in the sense that a failure of any one of them leads to dramatic, life-long consequences. Hence the importance of determining the sequential maturation of neurons, synapses and cortical maps. It is also important to determine how network-driven events become installed, as neuronal activity intervenes in all of these steps and modulates, for better or worse, the outcome. A fundamental consequence of these sequential events is that any disruption will have very different consequences depending on when it occurs, indeed, "when is as important as what". An obvious aspect of these general features is related to seizures. In fact, the developing brain has both a higher incidence of seizures in human and animal models, and experiences seizures that can produce long-lasting consequences that are also stage-dependent. This seminar and the series of slides presented are an introduction to these issues, summing up several studies made notably by INMED researchers during the last two decades (http://www.inmednet.com). It concentrates on four basic developmental rules: i) the generation by very immature neurons, of very large currents mediated by the activation of receptors in neurons that bear no synapses. This is due to the release of GABA that diffuses to distal sites and acts as a paracrine factor; ii) the excitatory/inhibitory shift of the actions of GABA during development because of a progressive reduction in the intracellular chloride concentration; iii) the sequential formation of GABAergic synapses and networks before glutamatergic ones, implying that, at an early stage, all the excitatory drive will be GABAergic; iv) the presence, at an early stage, of a unique, primitive pattern in all developing structures, this pattern disappears when most GABAergic synapses have shifted to their adult configuration. Several consequences of these sequences are described including: i) a control of neuronal migration by GABA-acting drugs, and the possibility that migration disorders are also generated by environmental factors that include the effects of GABA-acting agents; ii) If GABA excites immature neurons and inhibits adult one, then GABA-acting agents will also produce different effects on the mother and the embryo; iii) early brain oscillations are generated by the periphery and propagate centrally - notably to the sensory-motor cortex, suggesting that peripherally-generated movements may provide an important signal for the formation of cortical maps, in keeping with the importance of embryonic movements; iv) "seizures beget seizures" in the developing brain. This has now been shown in a triple chamber with the two intact hippocampi that we developed, and with which it has been possible to show that only recurrent seizures that include high frequency oscillations can transform the naïve, contralateral hippocampus to an epileptic one that seizes spontaneously. Most interestingly, at an early developmental stage, when GABA excites many neurons and the density of glutamatergic synapses is not sufficiently high, purely glutamatergic seizures cannot lead to long-term consequences, the additional excitatory drive provided by GABAergic synapses is needed. In other words, at that stage, blocking GABA synapses generates seizures, as in adults, but these do not lead to long-term consequences. The mechanisms that underlie these differences is due to the need for high frequency oscillations (> 80 Hz or so), and these can only be generated when GABA synapses are operative in the developing brain: GABA receptor antagonists are ictogenic, but not epileptogenic. To facilitate teaching purposes the paper is published together with supplemental data (as a PowerPoint presentation included in the accompanying DVD), thus allowing an overview of important developmental steps and their implications.

Mechanisms of rhythmogenesis: insights from hypothalamic vasopressin neurons.

Many neurons in the CNS, including hypothalamic vasopressin-expressing cells, display rhythmic activity patterning. These vasopressin neurons receive random synaptic input but fire action potentials in alternating periods of activity and silence that each lasts tens of seconds. Recent work demonstrates that vasopressin cell rhythmicity depends on feedback modulation of intrinsic membrane properties and synaptic inputs by peptides released from the dendrites of these neurons. Many other neurons across the CNS release neurotransmitters from their dendrites; therefore, vasopressin cells provide an insight into the potential mechanisms that support neuronal activity patterning across the CNS.

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep.

BACKGROUND: The gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA). METHODS: Antibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference. RESULTS: In adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01). CONCLUSION: Evidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep.

Constitutive activation of met kinase in non-small-cell lung carcinomas correlates with anchorage-independent cell survival.

Lung cancer is currently the most frequent cause of cancer death in North America. Hepatocyte growth factor (HGF) and its receptor Met are frequently over-expressed in non-small-cell lung carcinomas (NSCLC), but their potential role in tumor progression is not clearly known. To assess the role of HGF/Met signaling in lung carcinomas, we have examined the expression, activation status, and function of Met in NSCLC cell lines (n = 7), established from primary tumors or pleural fluids of cancer patients. We observed Met expression in three NSCLC cell lines, two of which exhibited constitutive tyrosine-phosphorylation of Met, and Met kinase activity. In addition, the observed constitutive activation of Met was sustained under anchorage-independent conditions, and correlated with phosphatidyl inositol 3-kinase-dependent cell survival. Immunoreactive HGF-like protein was secreted by two Met-positive and two Met-negative NSCLC cell lines. However HGF activity, as determined by the ability to induce cell scattering and tyrosine-phosphorylation of Met in reporter cell lines, was detected in conditioned medium from only one Met-negative NSCLC cell line: none of the conditioned media from Met-expressing NSCLC cell lines showed detectable HGF activity. Thus, constitutive activation of Met in NSCLC cell lines may occur at least in part through intracrine, or HGF-independent mechanisms. Interestingly, additional paracrine stimulation with exogenous recombinant HGF was required for DNA synthesis and correlated with increased activation of ERK1/2 in all Met-positive NSCLC cell lines, regardless of the basal activation status of Met. These findings indicate that a medium level of constitutive activation of Met occurs in some NSCLC cell lines, and correlates with survival of detached carcinoma cells; whereas additional paracrine stimulation by recombinant HGF is required for DNA synthesis. Thus constitutive and paracrine activation of Met may provide complementary signals that promote survival and proliferation, respectively, during tumor progression of NSCLC.

Complexity of the neuroendocrine system.

This paper summarizes briefly the numerous connections and mechanisms existing within the neuroendocrine system. In the last decades it became clear that besides the originally postulated basic mechanisms there are several other, such as autocrine and paracrine mechanisms within the glands, bidirectional neural connections between the target endocrine glands and the hypothalamus and lower brainstem, which are of functional significance. Further we learned that the organization of the neural structures involved is much more complicated than originally thought. In addition, it turned out that the neuroendocrine system and the immune system are closely and intimately linked to each other. The available informations indicate clearly that a physiological integration exists between the nervous, the endocrine and immune systems.