Oxidized/deamidated-ceruloplasmin dysregulates choroid plexus epithelial cells functionality and barrier properties via RGD-recognizing integrin binding.

Abstract Choroid plexus epithelial cells (CPEpiCs) determine the composition of cerebrospinal fluid (CSF) and constitute the blood-CSF barrier (BCSFB), functions that are altered in neurodegenerative diseases. In Parkinson's disease (PD) the pathological environment oxidizes and deamidates the ceruloplasmin, a CSF-resident ferroxidase, which undergoes a gain of RGD-recognizing integrin binding property, that may result in signal transduction. We investigated the effects that oxidized/deamidated ceruloplasmin (Cp-ox/de) may exert on CPEpiCs functions. Through RGD-recognizing integrins binding, Cp-ox/de mediates CPEpiCs adhesion and intracellular signaling, resulting in cell proliferation inhibition and alteration of the secretome profile in terms of proteins related to cell-extracellular matrix interaction. Oxidative conditions, comparable to those found in the CSF of PD patients, induced CPEpiCs barrier leakage, allowing Cp-ox/de to cross it, transducing integrins-mediated signal that further worsens BCSFB integrity. This mechanism might contribute to PD pathological processes altering CSF composition and aggravating the already compromised BCSFB function

An In Vitro Partial Lesion Model of Differentiated Human Mesencephalic Neurons : Effect of Pericyte Secretome on Phenotypic Markers

Parkinson’s disease (PD) is characterised by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta. Post-mortem data suggests that the loss of DA markers may long precede the cell death, leaving a window to rescue the DA phenotype. Screening for potential neuroprotective or restorative therapies, however, requires that partial lesions of DA neurons can be modelled in vitro. In order to establish a partial lesion model of DA neurons in vitro, we evaluated the effects of different exposure times to 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) on the cell survival and DA marker expression using DA neurons derived from the Lund human mesencephalic (LUHMES) cell line. We show that 24-h incubation with 50 μM of MPP+ or 6-h incubation with 100 μM of 6-OHDA leads to a significant decrease in the protein expression of DA markers without affecting overall cell death, consistent with a mild DA lesion. Using conditioned medium of human brain–derived pericytes stimulated with platelet-derived growth factor BB (PDGF-BB), we demonstrate a significant upregulation of DA markers. In conclusion, we provide an experimental model of an in vitro DA neuron partial lesion suitable to study different molecules and their potential neuroprotective or neurorestorative effects on the DA phenotype. We provide evidence that the secretome of brain pericytes stimulated via PDGF-BB/PDGFRβ affects DA marker expression and may represent one possible mechanism contributing to the neurorestoration previously observed in PD by this growth factor

The pericyte secretome : Potential impact on regeneration

Personalized and regenerative medicine is an emerging therapeutic strategy that is based on cell biology and biomedical engineering used to develop biological substitutes to maintain normal function or restore damaged tissues and organs. The secretory capacities of different cell types are now explored as such possible therapeutic regenerative agents in a variety of diseases. A secretome can comprise chemokines, cytokines, growth factors, but also extracellular matrix components, microvesicles and exosomes as well as genetic material and may differ depending on the tissue and the stimulus applied to the cell. With regard to clinical applications, the secretome of mesenchymal stem cells (MSC) is currently the most widely explored. However, other cell types such as pericytes may have similar properties as MSC and the potential therapeutic possibilities of these cells are only just beginning to emerge. In this review, we will summarize the currently available data describing the secretome of pericytes and its potential implications for tissue regeneration, whereby we especially focus on brain pericytes as potential new target cell for neuroregeneration and brain repair

Pericytes secrete pro-regenerative molecules in response to platelet-derived growth factor-BB

Brain pericytes not only maintain the anatomical, biochemical and immune blood-brain barrier, but display features of mesenchymal stem cells (MSCs) in vitro. MSCs have pro-regenerative properties attributed to their secretome. However, whether also brain pericytes possess such pro-regenerative capacities is largely unknown. Here we characterize the secretome and microvesicle (MV) release of human brain pericytes mediated by platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor beta (PDGFRβ) signalling. Upon PDGF-BB, pericytes release not only a plethora of growth factors and a panel of cytokines, but also MVs containing BDNF, FGFb, βNGF, VEGF and PLGF, a response that is specific for PDGFRβ signalling and activation of the ERK 1/2 pathway. In contrast, lipopolysaccharide (LPS), an activator of the innate immune system, stimulates the secretion of much higher amounts of mainly inflammatory cytokines and activates the NFκB pathway. Pericytes change their morphology and undergo opposite changes in surface marker expression, respectively. Our findings provide evidence that the secretome of human brain pericytes varies greatly depending on the exogenous stimulus. The differential secretory functions of pericytes may play an important role in either regulating neuroinflammation or contributing to neurorestoration and identify a possible new target cell for neuroregeneration

STAT3 precedes HIF1α transcriptional responses to oxygen and oxygen and glucose deprivation in human brain pericytes

Brain pericytes are important to maintain vascular integrity of the neurovascular unit under both physiological and ischemic conditions. Ischemic stroke is known to induce an inflammatory and hypoxic response due to the lack of oxygen and glucose in the brain tissue. How this early response to ischemia is molecularly regulated in pericytes is largely unknown and may be of importance for future therapeutic targets. Here we evaluate the transcriptional responses in in vitro cultured human brain pericytes after oxygen and/or glucose deprivation. Hypoxia has been widely known to stabilise the transcription factor hypoxia inducible factor 1-alpha (HIF1α) and mediate the induction of hypoxic transcriptional programs after ischemia. However, we find that the transcription factors Jun Proto-Oncogene (c-JUN), Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells (NFκB) and signal transducer and activator of transcription 3 (STAT3) bind genes regulated after 2hours (hs) of omitted glucose and oxygen before HIF1α. Potent HIF1α responses require 6hs of hypoxia to substantiate transcriptional regulation comparable to either c-JUN or STAT3. Phosphorylated STAT3 protein is at its highest after 5 min of oxygen and glucose (OGD) deprivation, whereas maximum HIF1α stabilisation requires 120 min. We show that STAT3 regulates angiogenic and metabolic pathways before HIF1α, suggesting that HIF1α is not the initiating trans-acting factor in the response of pericytes to ischemia

Platelet-derived growth factor-BB has neurorestorative effects and modulates the pericyte response in a partial 6-hydroxydopamine lesion mouse model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease where the degeneration of the nigrostriatal pathway leads to specific motor deficits. There is an unmet medical need for regenerative treatments that stop or reverse disease progression. Several growth factors have been investigated in clinical trials to restore the dopaminergic nigrostriatal pathway damaged in PD. Platelet-derived growth factor-BB (PDGF-BB), a molecule that recruits pericytes to stabilize microvessels, was recently investigated in a phase-1 clinical trial, showing a dose-dependent increase in dopamine transporter binding in the putamen of PD patients. Interestingly, evidence is accumulating that PD is paralleled by microvascular changes, however, whether PDGF-BB modifies pericytes in PD is not known. Using a pericyte reporter mouse strain, we investigate the functional and restorative effect of PDGF-BB in a partial 6-hydroxydopamine medial forebrain bundle lesion mouse model of PD, and whether this restorative effect is accompanied by changes in pericyte features. We demonstrate that a 2-week treatment with PDGF-BB leads to behavioural recovery using several behavioural tests, and partially restores the nigrostriatal pathway. Interestingly, we find that pericytes are activated in the striatum of PD lesioned mice and that these changes are reversed by PDGF-BB treatment. The modulation of brain pericytes may contribute to the PDGF-BB-induced neurorestorative effects, PDGF-BB allowing for vascular stabilization in PD. Pericytes might be a new cell target of interest for future regenerative therapies

The Effect of Aging on Nerve Morphology and Substance P Expression in Mouse and Human Corneas

Purpose Aging impairs corneal nerve density and sensitivity. Substance P (SP), a neuropeptide secreted by sensory nerves, regulates nerve morphology and nociception. Here, we investigate the relationship between aging, nerve morphology, and SP expression in mouse and human corneas. Methods SP levels in mouse corneas (wild type and substance P-knockout) and human corneas and tears were quantified with an ELISA assay. Corneal total nerve length (TNL) was measured with whole-mount β3-tubulin immunofluorescence in mouse and in vivo laser corneal confocal microscopy in humans. SP and β3-tubulin stained cross-sections were used to assess the colocalization of SP and nerves in human and mouse corneas. Ocular surface nociception was assessed with a wiping test in mice. Results SP colocalizes with sub-basal neurons in mice and humans. In WT mice, SP levels decrease with age (P = 0.0045, 8 vs. 52 weeks; P = 0.004, 26 vs. 52 weeks) as well as TNL (P = 0.018, 8 vs. 26 weeks; P = 0.0001, 8 vs. 52 weeks). Knockout mice show a greater TNL reduction (8 vs. 26 weeks, P = 0.0016) than WT mice. In the oldest WT and age-matched KO mice, nociception is impaired (P = 0.007 and P < 0.0001, respectively), and KO mice sensitivity is restored by topical SP treatment. In humans, SP levels are reduced in old subject corneas and correlate, in tears, with age (P = 0.0368); TNL also decreases in older patients (P = 0.0002). Conclusions Age-associated corneal nerve loss is paralleled by reduction of SP expression in mice and humans. SP promotes the maintenance of normal nerve morphology in the long term and modulates nociception in the cornea

Loss of Regulator of G-Protein Signaling 5 Leads to Neurovascular Protection in Stroke

Background and Purpose- In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Pericyte detachment contributes to BBB disruption and neurovascular dysfunction, but little is known about its regulation in stroke. Here, we investigated how loss of RGS5 (regulator of G protein signaling 5) in pericytes affects BBB breakdown in stroke and its consequences. Method- We used RGS5 knockout and control mice and applied a permanent middle cerebral occlusion model. We analyzed pericyte numbers, phenotype, and vessel morphology using immunohistochemistry and confocal microscopy. We investigated BBB breakdown by measuring endothelial coverage, tight junctions, and AQP4 (aquaporin 4) in addition to BBB permeability (fluorescent-conjugated dextran extravasation). Tissue hypoxia was assessed with pimonidazole hydrochloride and neuronal death quantified with the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results- We demonstrate that loss of RGS5 increases pericyte numbers and their endothelial coverage, which is associated with higher capillary density and length, and significantly less BBB damage after stroke. Loss of RGS5 in pericytes results in reduced vascular leakage and preserved tight junctions and AQP4, decreased cerebral hypoxia, and partial neuronal protection in the infarct area. Conclusions- Our findings show that loss of RGS5 affects pericyte-related BBB preservation in stroke and identifies RGS5 as an important target for neurovascular protection