Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function.

OBJECTIVE: The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques. METHODS: Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology. RESULTS: Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident. Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca2+ elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs. CONCLUSIONS: Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.

Short-term high-fat diet feeding of mice suppresses catecholamine-stimulated Ca2+ signalling in hepatocytes and intact liver.

Excess consumption of carbohydrates, fat and calories leads to non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance; these are major factors in the pathogenesis of type II diabetes. Hormones and catecholamines acting through G-protein coupled receptors (GPCRs) linked to phospholipase C (PLC) and increases in cytosolic Ca2+ ([Ca2+ ]c ) regulate many metabolic functions of the liver. In the intact liver, catabolic hormones such as glucagon, catecholamines and vasopressin integrate and synergize to regulate the frequency and extent to which [Ca2+ ]c waves propagate across hepatic lobules to control metabolism. Dysregulation of hepatic Ca2+ homeostasis has been implicated in the development of metabolic disease, but changes in hepatic GPCR-dependent Ca2+ signalling have been largely unexplored in this context. We show that short-term, 1-week, high-fat diet (HFD) feeding of mice attenuates noradrenaline-stimulated Ca2+ signalling, reducing the number of cells responding and suppressing the frequency of [Ca2+ ]c oscillations in both isolated hepatocytes and intact liver. The 1-week HFD feeding paradigm did not change basal Ca2+ homeostasis; endoplasmic reticulum Ca2+ load, store-operated Ca2+ entry and plasma membrane Ca2+ pump activity were unchanged compared to low-fat diet (LFD)-fed controls. However, noradrenaline-induced inositol 1,4,5-trisphosphate production was significantly reduced after HFD feeding, demonstrating an effect of HFD on receptor-stimulated PLC activity. Thus, we have identified a lesion in the PLC signalling pathway induced by short-term HFD feeding, which interferes with hormonal Ca2+ signalling in isolated hepatocytes and the intact liver. These early events may drive adaptive changes in signalling, which lead to pathological consequences in fatty liver disease. KEY POINTS: Non-alcoholic fatty liver disease (NAFLD) is a growing epidemic. In healthy liver, the counteracting effects of catabolic and anabolic hormones regulate metabolism and energy storage as fat. Hormones and catecholamines promote catabolic metabolism via increases in cytosolic Ca2+ ([Ca2+ ]c ). We show that 1 week high-fat diet (HFD) feeding of mice attenuated the Ca2+ signals induced by physiological concentrations of noradrenaline. Specifically, HFD suppressed the normal pattern of periodic [Ca2+ ]c oscillations in isolated hepatocytes and disrupted the propagation of intralobular [Ca2+ ]c waves in the intact perfused liver. Short-term HFD inhibited noradrenaline-induced inositol 1,4,5-trisphosphate generation, but did not change basal endoplasmic reticulum Ca2+ load or plasma membrane Ca2+ fluxes. We propose that impaired Ca2+ signalling plays a key role in the earliest phases of the etiology of NAFLD, and is responsible for many of the ensuing metabolic and related dysfunctional outcomes at the cellular and whole tissue level.

Fmr1 exon 14 skipping in late embryonic development of the rat forebrain.

BACKGROUND: Fragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets. RESULTS: We observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17-E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A. CONCLUSIONS: The forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene's expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.

ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington's disease: a novel approach for cell clock research.

Calcium, the most versatile second messenger, regulates essential biology including crucial cellular events in embryogenesis. We investigated impacts of calcium channels and purinoceptors on neuronal differentiation of normal mouse embryonic stem cells (ESCs), with outcomes being compared to those of in vitro models of Huntington's disease (HD). Intracellular calcium oscillations tracked via real-time fluorescence and luminescence microscopy revealed a significant correlation between calcium transient activity and rhythmic proneuronal transcription factor expression in ESCs stably expressing ASCL-1 or neurogenin-2 promoters fused to luciferase reporter genes. We uncovered that pharmacological manipulation of L-type voltage-gated calcium channels (VGCCs) and purinoceptors induced a two-step process of neuronal differentiation. Specifically, L-type calcium channel-mediated augmentation of spike-like calcium oscillations first promoted stable expression of ASCL-1 in differentiating ESCs, which following P2Y2 purinoceptor activation matured into GABAergic neurons. By contrast, there was neither spike-like calcium oscillations nor responsive P2Y2 receptors in HD-modeling stem cells in vitro. The data shed new light on mechanisms underlying neurogenesis of inhibitory neurons. Moreover, our approach may be tailored to identify pathogenic triggers of other developmental neurological disorders for devising targeted therapies.

A Tale of two receptors.

Calcium (Ca2+) oscillations in hepatocytes have a wide dynamic range. In particular, recent experimental evidence shows that agonist stimulation of the P2Y family of receptors leads to qualitatively diverse Ca2+ oscillations. We present a new model of Ca2+ oscillations in hepatocytes based on these experiments to investigate the mechanisms controlling P2Y-activated Ca2+ oscillations. The model accounts for Ca2+ regulation of the IP3 receptor (IP3R), the positive feedback from Ca2+ on phospholipase C (PLC) and the P2Y receptor phosphorylation by protein kinase C (PKC). Furthermore, PKC is shown to control multiple cellular substrates. Utilising the model, we suggest the activity and intensity of PLC and PKC necessary to explain the qualitatively diverse Ca2+ oscillations in response to P2Y receptor activation.

Rare CACNA1H and RELN variants interact through mTORC1 pathway in oligogenic autism spectrum disorder.

Oligogenic inheritance of autism spectrum disorder (ASD) has been supported by several studies. However, little is known about how the risk variants interact and converge on causative neurobiological pathways. We identified in an ASD proband deleterious compound heterozygous missense variants in the Reelin (RELN) gene, and a de novo splicing variant in the Cav3.2 calcium channel (CACNA1H) gene. Here, by using iPSC-derived neural progenitor cells (NPCs) and a heterologous expression system, we show that the variant in Cav3.2 leads to increased calcium influx into cells, which overactivates mTORC1 pathway and, consequently, further exacerbates the impairment of Reelin signaling. Also, we show that Cav3.2/mTORC1 overactivation induces proliferation of NPCs and that both mutant Cav3.2 and Reelin cause abnormal migration of these cells. Finally, analysis of the sequencing data from two ASD cohorts-a Brazilian cohort of 861 samples, 291 with ASD; the MSSNG cohort of 11,181 samples, 5,102 with ASD-revealed that the co-occurrence of risk variants in both alleles of Reelin pathway genes and in one allele of calcium channel genes confer significant liability for ASD. Our results support the notion that genes with co-occurring deleterious variants tend to have interconnected pathways underlying oligogenic forms of ASD.

Effects of Magnetite Nanoparticles and Static Magnetic Field on Neural Differentiation of Pluripotent Stem Cells.

Neurodevelopmental processes of pluripotent cells, such as proliferation and differentiation, are influenced by external natural forces. Despite the presence of biogenic magnetite nanoparticles in the central nervous system and constant exposure to the Earth's magnetic fields and other sources, there is scant knowledge regarding the role of electromagnetic stimuli in neurogenesis. Moreover, emerging applications of electrical and magnetic stimulation to treat neurological disorders emphasize the relevance of understanding the impact and mechanisms behind these stimuli. Here, the effects of magnetic nanoparticles (MNPs) in polymeric coatings and the static external magnetic field (EMF) were investigated on neural induction of murine embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs). The results show that the presence of 0.5% MNPs in collagen-based coatings facilitates the migration and neuronal maturation of mESCs and hiPSCs in vitro. Furthermore, the application of 0.4 Tesla EMF perpendicularly to the cell culture plane, discernibly stimulates proliferation and guide fate decisions of the pluripotent stem cells, depending on the origin of stem cells and their developmental stage. Mechanistic analysis reveals that modulation of ionic homeostasis and the expression of proteins involved in cytostructural, liposomal and cell cycle checkpoint functions provide a principal underpinning for the impact of electromagnetic stimuli on neural lineage specification and proliferation. These findings not only explore the potential of the magnetic stimuli as neural differentiation and function modulator but also highlight the risks that immoderate magnetic stimulation may affect more susceptible neurons, such as dopaminergic neurons.

Receptor-specific Ca2+ oscillation patterns mediated by differential regulation of P2Y purinergic receptors in rat hepatocytes.

Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.

Effects of single-dose antipurinergic therapy on behavioral and molecular alterations in the valproic acid-induced animal model of autism.

Autism spectrum disorder (ASD) is characterized by deficits in communication and social interaction, restricted interests, and stereotyped behavior. Environmental factors, such as prenatal exposure to valproic acid (VPA), may contribute to the increased risk of ASD. Since disturbed functioning of the purinergic signaling system has been associated with the onset of ASD and used as a potential therapeutic target for ASD in both clinical and preclinical studies, we analyzed the effects of suramin, a non-selective purinergic antagonist, on behavioral, molecular and immunological in an animal model of autism induced by prenatal exposure to VPA. Treatment with suramin (20 mg/kg, intraperitoneal) restored sociability in the three-chamber apparatus and decreased anxiety measured by elevated plus maze apparatus, but had no impact on decreased reciprocal social interactions or higher nociceptive threshold in VPA rats. Suramin treatment did not affect VPA-induced upregulation of P2X4 and P2Y2 receptor expression in the hippocampus, and P2X4 receptor expression in the medial prefrontal cortex, but normalized an increased level of interleukin 6 (IL-6). Our results suggest an important role of purinergic signaling modulation in behavioral, molecular, and immunological aberrations described in VPA model, and indicate that the purinergic signaling system might be a potential target for pharmacotherapy in preclinical studies of ASD.

P2Y6 and P2X7 Receptor Antagonism Exerts Neuroprotective/ Neuroregenerative Effects in an Animal Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by decreased dopamine bioavailability in the substantia nigra and the striatum. Taking into account that adenosine-5'-triphosphate (ATP) and its metabolites are intensely released in the 6-hydroxydopamine (6-OHDA) animal model of PD, screening of purinergic receptor gene expression was performed. Effects of pharmacological P2Y6 or P2X7 receptor antagonism were studied in preventing or reversing hemiparkinsonian behavior and dopaminergic deficits in this animal model. P2X7 receptor antagonism with Brilliant Blue G (BBG) at a dose of 75 mg/kg re-established the dopaminergic nigrostriatal pathway in rats injured with 6-OHDA. Selective P2Y6 receptor antagonism by MRS2578 prevented dopaminergic neuron death in SH-SY5Y cells in vitro and in vivo in the substantia nigra of rats injured with 6-OHDA. Moreover, in vivo analysis showed that both treatments were accompanied by a reduction of microglial activation in the substantia nigra. Altogether, these data provide evidence that antagonism of P2X7 or P2Y6 receptors results in neuroregenerative or neuroprotective effects, respectively, possibly through modulation of neuroinflammatory responses.

Pathophysiology in the comorbidity of Bipolar Disorder and Alzheimer's Disease: pharmacological and stem cell approaches.

Neuropsychiatric disorders involve various pathological mechanisms, resulting in neurodegeneration and brain atrophy. Neurodevelopmental processes have shown to be critical for the progression of those disorders, which are based on genetic and epigenetic mechanisms as well as on extrinsic factors. We review here common mechanisms underlying the comorbidity of Bipolar Disorders and Alzheimer's Disease, such as aberrant neurogenesis and neurotoxicity, reporting current therapeutic approaches. The understanding of these mechanisms precedes stem cell-based strategies as a new therapeutic possibility for treatment and prevention of Bipolar and Alzheimer's Disease progression. Taking into account the difficulty of studying the molecular basis of disease progression directly in patients, we also discuss the importance of stem cells for effective drug screening, modeling and treating psychiatric diseases, once in vitro differentiation of patient-induced pluripotent stem cells provides relevant information about embryonic origins, intracellular pathways and molecular mechanisms.

Purinergic Receptors in Neurological Diseases With Motor Symptoms: Targets for Therapy.

Since proving adenosine triphosphate (ATP) functions as a neurotransmitter in neuron/glia interactions, the purinergic system has been more intensely studied within the scope of the central nervous system. In neurological disorders with associated motor symptoms, including Parkinson's disease (PD), motor neuron diseases (MND), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's Disease (HD), restless leg syndrome (RLS), and ataxias, alterations in purinergic receptor expression and activity have been noted, indicating a potential role for this system in disease etiology and progression. In neurodegenerative conditions, neural cell death provokes extensive ATP release and alters calcium signaling through purinergic receptor modulation. Consequently, neuroinflammatory responses, excitotoxicity and apoptosis are directly or indirectly induced. This review analyzes currently available data, which suggests involvement of the purinergic system in neuro-associated motor dysfunctions and underlying mechanisms. Possible targets for pharmacological interventions are also discussed.

P2Y12 but not P2Y13 Purinergic Receptor Controls Postnatal Rat Retinogenesis In Vivo.

Adenine nucleotides through P2Y1 receptor stimulation are known to control retinal progenitor cell (RPC) proliferation by modulating expression of the p57KIP2, a cell cycle regulator. However, the role of Gi protein-coupled P2Y12 and P2Y13 receptors also activated by adenine nucleotides in RPC proliferation is still unknown. Gene expression of the purinergic P2Y12 subtype was detected in rat retina during early postnatal days (P0 to P5), while expression levels of P2Y13 were low. Immunohistochemistry assays performed with rat retina on P3 revealed P2Y12 receptor expression in both Ki-67-positive cells in the neuroblastic layer and Ki-67-negative cells in the ganglion cell layer and inner nuclear layer. Nonetheless, P2Y13 receptor expression could not be detected in any stratum of rat retina. Intravitreal injection of PSB 0739 or clopidogrel, both selective P2Y12 receptor antagonists, increased by 20 and 15%, respectively, the number of Ki-67-positive cells following 24 h of exposure. Moreover, the P2Y12 receptor inhibition increased cyclin D1 and decreased p57KIP2 expression. However, there were no changes in the S phase of the cell cycle (BrdU-positive cells) or in mitosis (phospho-histone-H3-positive cells). Interestingly, an increase in the number of cyclin D1/TUNEL-positive cells after treatment with PSB 0739 was observed. These data suggest that activation of P2Y12 receptors is required for the successful exit of RPCs from cell cycle in the postnatal rat retina.

Kinin and Purine Signaling Contributes to Neuroblastoma Metastasis.

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.