Corneal and Epidermal Nerve Quantification in Chemotherapy Induced Peripheral Neuropathy.

Chemotherapy-induced neurotoxicity is an increasingly recognized clinical issue in oncology. in vivo confocal microscopy (IVCM) of corneal nerves has been successfully used to diagnose peripheral neuropathies, including diabetic neuropathy. The purpose of this study was to test if the combination of corneal nerve density and morphology assessed by IVCM is useful to monitor the neurotoxic effects of chemotherapy compared to epidermal nerve quantification. Overall, 95 adult patients with different cancer types were recruited from the oncology and hematology departments of the San Raffaele Hospital. Neurological examination, including clinical Total Neuropathy Score, and in vivo corneal confocal microscopy (IVCM), were performed before and after chemotherapy. In a group of 14 patients, skin biopsy was performed at the first and last visit. In the group of 14 patients who underwent both skin biopsy and corneal nerve imaging, clinical worsening (+69%, p = 0.0018) was paralleled by corneal nerve fiber (CNF) density reduction (-22%, p = 0.0457). Clinical Total neuropathy score significantly worsened from the first to the last visit (+62%, p < 0.0001). CNF length was not significantly reduced overall. However, CNF density/tortuosity ratio significantly decreased after therapy. Correlation analysis showed that the CNF density/tortuosity ratio was also correlated with the number of chemotherapy cycles (r = -0.04790, P = 0.0009). Our data confirm that in vivo corneal confocal microscopy is a helpful, non-invasive tool which shows promise for the diagnosis of chemotherapy-induced peripheral neuropathies. IVCM could allow a rapid, reproducible and non-invasive quantification of peripheral nerve pathology in chemotherapy-associated neuropathy.

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

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.

Ceruloplasmin oxidized and deamidated by Parkinson's disease cerebrospinal fluid induces epithelial cells proliferation arrest and apoptosis.

In Parkinson's disease, the ferroxidase ceruloplasmin (Cp) is oxidized and deamidated by the pathological cerebrospinal fluid (CSF) environment. These modifications promote the gain of integrin binding properties, fostered by the deamidation of two NGR-motifs present in the Cp sequence that convert into the isoDGR-motif. Through isoDGR/integrin binding, the oxidized/deamidated-Cp (Cp-ox/de) mediates cell adhesion and transduces an intracellular signal in epithelial cells that seems to be addressed to regulate cell cycle, proliferation and cytoskeletal re-arrangement. However, the effect fostered on cells by integrins engagement via Cp-ox/de is not known. We found that in HaCaT epithelial cells, the incubation with Cp-ox/de resulted in proliferation inhibition mediated by isoDGR, cell cycle arrest and apoptosis induction. Similar proliferation inhibition was induced by treatment with purified Cp previously incubated in the CSF from Parkinson's disease patients, but not by Cp incubated in the CSF from healthy subjects. In human primary choroid plexus epithelial cells, a possible in vivo target of Cp-ox/de generated in pathological CSFs, we found that Cp-ox/de mediated cell adhesion via isoDGR/integrins binding and transduced an intracellular signal, which resulted in cell proliferation inhibition. Thus, the generation of Cp-ox/de in pathological CSFs and the consequent apoptosis induction of epithelial cells facing the liquor, might represent a novel mechanism that contributes to neurodegeneration.

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.

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.

Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients.

A humoral immune response against aberrant tumor proteins can be elicited in cancer patients, resulting in the production of auto-antibodies (Abs). By serological proteome analysis we identified the surface membrane protein ADAM10, a metalloproteinase that has a role in epithelial-tumor progression and invasion, as a target of the immune response in colorectal cancer (Crc). A screening carried out on the purified protein using testing cohorts of sera (Crc patients n = 57; control subjects n = 39) and validation cohorts of sera (Crc patients n = 49; control subjects n = 52) indicated that anti-ADAM10 auto-Abs were significantly induced in a large group (74%) of colon cancer patients, in particular in patients at stage II and III of the disease. Interestingly, in Crc patients classified as stage III disease, the presence of anti-ADAM10 auto-Abs in the sera was associated with a favourable follow-up with a significant shifting of the recurrence-free survival median time from 23 to 55 months. Even though the ADAM10 protein was expressed in Crc regardless the presence of auto-Abs, the immature/non-functional isoform of ADAM10 was highly expressed in the tumor of anti-ADAM10-positive patients and was the isoform targeted by the auto-Abs. In conclusion, the presence of anti-ADAM10 auto-Abs seems to reflect the increased tumor expression of the immunogenic immature-ADAM10 in a group of Crc patients, and is associated with a favourable prognosis in patients at stage III of the disease.

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.

Oxidation-induced structural changes of ceruloplasmin foster NGR motif deamidation that promotes integrin binding and signaling.

Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface ((568)NGR) and the other buried in the tertiary structure ((962)NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed (568)NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden (962)NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion.

Ceruloplasmin oxidation, a feature of Parkinson's disease CSF, inhibits ferroxidase activity and promotes cellular iron retention.

Parkinson's disease is a neurodegenerative disorder characterized by oxidative stress and CNS iron deposition. Ceruloplasmin is an extracellular ferroxidase that regulates cellular iron loading and export, and hence protects tissues from oxidative damage. Using two-dimensional electrophoresis, we investigated ceruloplasmin patterns in the CSF of human Parkinson's disease patients. Parkinson's disease ceruloplasmin profiles proved more acidic than those found in healthy controls and in other human neurological diseases (peripheral neuropathies, amyotrophic lateral sclerosis, and Alzheimer's disease); degrees of acidity correlated with patients' pathological grading. Applying an unsupervised pattern recognition procedure to the two-dimensional electrophoresis images, we identified representative pathological clusters. In vitro oxidation of CSF in two-dimensional electrophoresis generated a ceruloplasmin shift resembling that observed in Parkinson's disease and co-occurred with an increase in protein carbonylation. Likewise, increased protein carbonylation was observed in Parkinson's disease CSF, and the same modification was directly identified in these samples on ceruloplasmin. These results indicate that ceruloplasmin oxidation contributes to pattern modification in Parkinson's disease. From the functional point of view, ceruloplasmin oxidation caused a decrease in ferroxidase activity, which in turn promotes intracellular iron retention in neuronal cell lines as well as in primary neurons, which are more sensitive to iron accumulation. Accordingly, the presence of oxidized ceruloplasmin in Parkinson's disease CSF might be used as a marker for oxidative damage and might provide new insights into the underlying pathological mechanisms.