Brain microvessels and neuronal excitability: who is exciting who?

The working hypothesis is that any stimulus acting on brain microvascular endothelium, might activate the neurovascular unit by intercellular crosstalk, and consequently could increase neuronal hyperexcitability. The first chapter presents the concept of neurovascular unit. Interactions between neurovascular unit components are also discussed, and their involvement in ictogenesis. The second chapter describes the aim of the thesis, the main objectives and the principal technical approaches used in the study. The third chapter is dedicated to the elusive interaction between Qtracker\uae800 vascular labels and brain endothelial microvascular endothelial cells. Results: (1) non-targeted PEGylated near-infrared emitting Qtracker\uae800 accumulate in brain vascular endothelium, (2) Qtracker\uae800 alters \u2018normal\u2019 calcium signalling in brain endothelial cells, (3) there are substantial inter-individual differences in human endothelial cells activation upon exposure to Qtracker\uae800, (4) Qtracker\uae800 may not be suitable for translational studies. In conclusion, although brain vasculature imaging techniques can greatly benefit from the use of nanoparticle labels, such labels may be internalized by and functionally interact with blood vessel endothelia, which raises obvious safety concerns. The fourth chapter makes a functional description of the muscarinic acetylcholine receptors in brain microvascular endothelial cells. Results: (1) all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells, (2) acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors, (3) The relative mRNA expression of M2-M5 correlates with their relative protein abundance, but a mismatch exists for M1 mRNA versus protein levels, (4) although M1 and M3 are the most abundant receptors, only a small fraction of the M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling, (5) bioinformatic analysis performed on eucaryotic species demonstrate the high degree of conservation of the orthosteric binding site and the great variability of the allosteric site, (6) muscarinic acetylcholine receptors represent potential pharmacological targets in future translational studies. In conclusion, our findings indicate that investigators should particularly focus on the allosteric binding sites of the M1 and M3 receptors. The fifth chapter describes molecular mechanisms of pilocarpine action at the level of brain microvascular endothelium. Results: (1) pilocarpine induces the in vivo and in vitro increase of the cytokines levels, (2) pilocarpine upregulates the expression of adhesion molecules in brain microvascular endothelial cells, (3) pilocarpine elicits calcium transients in brain microvascular endothelial cells but is not inducing epileptic-like discharges in hippocampal pyramidal neurons, (4) pilocarpine downregulates the expression of tight junction proteins and permeabilizes the monolayers of brain microvascular endothelial cells, (5) pilocarpine competes with acetylcholine on the same binding site of the muscarinic receptors and regulates their expression. In conclusion, our study indicates that brain endothelium is an important site of action for pilocarpine, and that neurons\u2019 exposure to pilocarpine is not triggering seizure-like activity, therefore epileptogenesis mechanisms should be revisited

Unleashing the Potential of Brain Endothelial Cells in Epilepsy

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Diabetic Neuropathy: Promises and Disappointments from Benchside to Bedside

Diabetic neuropathy is the most common complication of diabetes and according to World Health Organization (WHO) almost 50% of diabetes affected patients have same degree of neuropathy [1]. In terms of health care costs, diabetic peripheral neuropathy has a high post-diagnosis economic impact being associated with a 20% increase in the number of patients visiting hospitals and a 46% increase in the number of visits to hospitals compared to the pre-diagnosis situation [2]. As diabetic neuropathy predisposes to the diabetic foot disease, it should be also considered as one of the major causes determining the lower limb amputation. In fact, diabetic neuropathy increases by 7-fold the risk of foot ulceration [3]. Diabetic neuropathy is associated with nerve damage and should be considered as an important factor contributing to disability, sexual dysfunction, urinary tract infections, digestive problems, social isolation, and depression [4]. Last by not least, diabetes increases by 2-4 fold the risk of stroke [5]. Therefore, social costs imply beside the effective health care costs for diabetes treatment, an additional budget for the social security disability, impotence treatment, depression treatment etc. Considering the variety of aspects, from health to economical ones, it is imperative to find new solutions (e.g. molecules/ markers) for early diagnosis of diabetes/ diabetic neuropathy and a concerted effort of the public and private funding should be focused to the benchside

Alterations in Calcium Signaling Pathways in Breast Cancer

Breast cancer is the second most common cancer in women and the fifth cause contributing to death due to the cancer condition. It is essential to deeply understand the complex cellular mechanisms leading to this disease. There are multiple connections between calcium homeostasis alterations and breast cancer in the literature, but no consensus links the mechanism to the disease prognosis. Among the cells contributing to the breast cancer are the breast telocytes, which connect through gap junctions to other cells, including cancer cells and myoepithelial cells. Multiple proteins (i.e., voltage-gated calcium channels, transient receptor potential channels, STIM and Orai proteins, ether à go-go potassium channels, calcium-activated potassium channels, calcium-activated chloride channels, muscarinic acetylcholine receptors, etc.) coupled with calcium signaling pathways undergo functional and/or expression changes associated with breast cancer development and progression, and might represent promising pharmacological targets. Unraveling the mechanisms of altered calcium homeostasis in various breast cells due to the cancer condition might contribute to personalized therapeutic approaches

Calcium signaling in interstitial cells: focus on telocytes

In this review, we describe the current knowledge on calcium signaling pathways in interstitial cells with a special focus on interstitial cells of Cajal (ICCs), interstitial Cajal-like cells (ICLCs), and telocytes. In detail, we present the generation of Ca(2+) oscillations, the inositol triphosphate (IP\u2083)/Ca(2+) signaling pathway and modulation exerted by cytokines and vasoactive agents on calcium signaling in interstitial cells. We discuss the physiology and alterations of calcium signaling in interstitial cells, and in particular in telocytes. We describe the physiological contribution of calcium signaling in interstitial cells to the pacemaking activity (e.g., intestinal, urinary, uterine or vascular pacemaking activity) and to the reproductive function. We also present the pathological contribution of calcium signaling in interstitial cells to the aortic valve calcification or intestinal inflammation. Moreover, we summarize the current knowledge of the role played by calcium signaling in telocytes in the uterine, cardiac and urinary physiology, and also in various pathologies, including immune response, uterine and cardiac pathologies

Extracellular Vesicles in Cancer

Extracellular vesicles (EVs) represent a generic term for all the secreted vesicles, which include exosomes, microvesicles, and apoptotic bodies. EVs are key partners in the intercellular communication and play an essential role in multiple physiological and pathological conditions. EVs are shuttles for cargo molecules, such as RNA (mRNA, microRNA, and other noncoding RNAs), DNA, proteins (receptors, transcription factors, enzymes, and extracellular matrix proteins), and lipids. In pathological states, including cancer, EVs might represent either useful biomarkers or can be used for therapeutic purposes. Moreover, in cancer, it was demonstrated that EVs play an essential role in drug resistance. Here, we review the role played by EVs in the most common forms of cancer, with a special focus on ovarian and breast cancers

Endothelial cells are key-players in pilocarpine-induced epileptogenesis

In recent years, the concept of the neurovascular unit (NVU) has emerged as a new paradigm for investigating both physiology and pathology in the CNS. This concept proposes that a purely neurocentric focus is not sufficient, and emphasizes that all cell types in the brain including neuronal, glial and vascular components (endothelial cells, blood cells, including immunity cells) must be examined in an integrated context. Cell–cell signaling and coupling between these different compartments thus form the basis for normal function (Lok et al. 2007). We tested the hypothesis that disordered signaling and perturbed coupling of these different components can be the basis for epileptogenesis in the pilocarpine model of epilepsy. We thus determined that pilocarpine can act on endothelial cells via receptors, comparing the response of the same stimulation in neurons as well

The effect of glucosamine, chondroitin and harpagophytum procumbens on femoral hyaline cartilage thickness in patients with knee osteoarthritis– An MRI versus ultrasonography study

Background: the evaluation of cartilage thickness has become possible with new techniques such as musculoskeletal ultrasonography (US) and magnetic resonance imagining (MRI), making the evaluation of the treatment response and the progression of the disease more accurate. Objective: to evaluate the efficacy of a Symptomatic Slow Acting Drug for Osteoarthritis using both US and MRI for measuring cartilage thickness at baseline and after 1 year. Methods: The study included the clinical evaluation of 20 patients at baseline, at 6 and 12 months as well as imaging exams (US and MRI) at baseline and after 1 year. Measurements were performed in both knees, in lateral and medial condyles, and in the intercondylar area. After the baseline visit, patients underwent a SYSADOA treatment which included Harpagophytum procumbens (HPc) administered on a daily basis, in a specific regimen. Results and discussions: The US examination permitted the detailed evaluation of the femoral hyaline cartilage thickness, with statistically significant differences before and after treatment at the level of the medial compartment, both in the dominant (1.59±0.49 vs. 1.68±0.49, p=0.0013) and non-dominant knee (1.73±0.53 vs. 1.79±0.52, p=0.0106). The US and the MRI correlated well (r=0.63) and showed no radiographic progression in knee osteoarthritis after one year of treatment with specific SYSADOA. Moreover, the US showed improvement in the cartilage thickness of the medial compartment. Conclusions: The combination with HPc could increase the delay in the radiographic progression of the knee osteoarthritis, with improvement of femoral hyaline cartilage thickness in the medial and lateral compartment. The US might be an important tool in OA evaluation and monitoring

Are they in or out? The elusive interaction between Qtracker(\uae)800 vascular labels and brain endothelial cells

AIM:Qtracker\uae800 Vascular labels (Qtracker\uae800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown.MATERIALS & METHODS:Qtracker\uae800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker\uae800 exposure on mouse and human endothelial cells by calcium imaging.RESULTS:Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker\uae800.CONCLUSION:Qtracker\uae800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging

All muscarinic acetylcholine receptors (M1-M5) are expressed in murine brain microvascular endothelium

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca(2+) signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors