Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Here, we show that extracellular HMGB1 and its receptor for advanced glycation end products (RAGE) induce both migration and proliferation of vessel-associated stem cells (mesoangioblasts), and thus may play a role in muscle tissue regeneration. In vitro, HMGB1 induces migration and proliferation of both adult and embryonic mesoangioblasts, and disrupts the barrier function of endothelial monolayers. In living mice, mesoangioblasts injected into the femoral artery migrate close to HMGB1-loaded heparin-Sepharose beads implanted in healthy muscle, but are unresponsive to control beads. Interestingly, α-sarcoglycan null dystrophic muscle contains elevated levels of HMGB1; however, mesoangioblasts migrate into dystrophic muscle even if their RAGE receptor is disabled. This implies that the HMGB1–RAGE interaction is sufficient, but not necessary, for mesoangioblast homing; a different pathway might coexist. Although the role of endogenous HMGB1 in the reconstruction of dystrophic muscle remains to be clarified, injected HMGB1 may be used to promote tissue regeneration

Isolation and characterization of mesoangioblasts from mouse, dog, and human tissues.

Mesoangioblasts are recently identified stem/progenitor cells, associated with small vessels of the mesoderm in mammals. Originally described in the mouse embryonic dorsal aorta, similar though not identical cells have been later identified and characterized from postnatal small vessels of skeletal muscle and heart (not described in this unit). They have in common the anatomical location, the expression of endothelial and/or pericyte markers, the ability to proliferate in culture, and the ability to undergo differentiation into various types of mesoderm cells upon proper culture conditions. Currently, the developmental origin of mesoangioblasts, their phenotypic heterogeneity, and the relationship with other mesoderm stem cells are not understood in detail and are the subject of active research. However, from a practical point of view, these cells have been successfully used in cell transplantation protocols that have yielded a significant rescue of structure and function in skeletal muscle of dystrophic mice and dogs. Since the corresponding human cells have been recently isolated and characterized, a clinical trial with these cells is planned in the near future. This unit provides detailed methods for isolation, culture, and characterization of mesoangioblasts

EFFETTO IPOTENSIVO E BRADICARDIZZANTE PROLUNGATO INDOTTO DALL\u27IPERESTENSIONE DEI MUSCOLI MANDIBOLARI: EVIDENZE PRELIMINARI NEL SOGGETTO VOLONTARIO

Introduction: The "trigemino-cardiac reflex" TCR has been known since the middle of the nineteenth century and consists in a decrease of heart rate (HR) and arterial blood pressure (BP) secondary to afferent trigeminal stimulation. This reflex is relatively poorly studied, except in anesthesiology, where it is known as a potential complication in cranio-facial surgery. Materials and Methods: We assessed in 20 normal volunteers (6M, 14F; 24-26 yrs) if a TCR can be elicited by means of trigeminal afferent neuromuscular spindle stimulation induced by 10 min mandibular stretching (obtained by an ad-hoc developed spring device). BP and HR were recorded intermittently by an OMRON M4 device up to 80 min (all subjects) and continuously by Finapres up to 15 min (n=9) after stretching. The following control procedures were employed: no treatment (n=20), 10 min gum chewing (n=9) and 10 min of keeping an ice-cream stick between the teeth (n=9). Results: Mandibular stretching was well tolerated and induced significant reductions (compared to pre-stretching) of systolic BP from 5 min onwards and of diastolic BP and HR from 15 min onwards, that lasted up to 80 min. No significant effects were observed in controls. Discussion: These observations indicate that mandibular stretching induces prolonged hypotensive and bradycardic effects, probably through a TCR, that may be of clinical and therapeutic significance.Introduzione: Gi? dalla met? dell\u27ottocento ? stato descritto il riflesso "trigemino-cardiaco" (RTC) come riduzione della pressione arteriosa (PA) e frequenza cardiaca (FC) in seguito a stimolazione delle branche sensoriali afferenti del trigemino. Tale riflesso ? stato relativamente poco studiato, salvo che in anestesiologia, quale complicanza di interventi di chirurgia maxillo-faciale. Materiali e Metodiche: Per valutare se lo stiramento dei fusi neuromuscolari di muscoli innervati dal trigemino possa attivare il RTC, ? stata misurata PA e FC in 20 volontari (6M, 14F, 24-26 aa) durante e dopo iperestensione mandibolare forzata per 10 min con una lamina di metallo piegata ad ansa e sviluppata ad hoc. Le misure sono state eseguite con registratore automatico (Omron M4) fino a 80 min dopo la prova in tutti i soggetti e con Finapres fino a 15 min dopo la prova, in 9 soggetti. Come controllo ? stato utilizzato: nessun trattamento (n=20), masticazione di chewing gum (n=9) e mantenimento di stecco di gelato tra i denti (n=9). Risultati: L\u27iperestensione forzata della mandibola ? stata ben tollerata e ha indotto una significativa riduzione (rispetto al basale: pre-estensione) della PA sistolica da 5 min e della PA diastolica e FC da 15 min dopo la prova. Tali effetti erano persistenti a 80 min. Nessun effetto significativo si ? osservato nei controlli. Discussione: Queste osservazioni evidenziano l\u27esistenza di un RTC in seguito a iperestensione mandibolare, che appare permanere a lungo dopo lo stimolo. Ci? potrebbe avere interessanti risvolti terapeutici

Alteration of the late endocytic pathway in Charcot-Marie-Tooth type 2B disease

The small GTPase RAB7A regulates late stages of the endocytic pathway and plays specific roles in neurons, controlling neurotrophins trafficking and signaling, neurite outgrowth and neuronal migration. Mutations in the RAB7A gene cause the autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) disease, an axonal peripheral neuropathy. As several neurodegenerative diseases are caused by alterations of endocytosis, we investigated whether CMT2B-causing mutations correlate with changes in this process. To this purpose, we studied the endocytic pathway in skin fibroblasts from healthy and CMT2B individuals. We found higher expression of late endocytic proteins in CMT2B cells compared to control cells, as well as higher activity of cathepsins and higher receptor degradation activity. Consistently, we observed an increased number of lysosomes, accompanied by higher lysosomal degradative activity in CMT2B cells. Furthermore, we found increased migration and increased RAC1 and MMP-2 activation in CMT2B compared to control cells. To validate these data, we obtained sensory neurons from patient and control iPS cells, to confirm increased lysosomal protein expression and lysosomal activity in CMT2B-derived neurons. Altogether, these results demonstrate that in CMT2B patient-derived cells, the endocytic degradative pathway is altered, suggesting that higher lysosomal activity contributes to neurodegeneration occurring in CMT2B.Peer reviewe

Raw data for Figure 4A: Neutralising antibodies against IFN-γ and TNF-α reduce the immunosuppressive capacity of Mesoangioblasts/HIDEMs

<p>CFSE labelled PBMCs were stimulated with anti-CD3/CD28 beads in the presence of HIDEMs/mesoangioblasts (1:4) and neutralising antibodies against IFN-γ and TNF-α or irrelevant isotype control antibody (0.5, 1.0 and 2.0 µg/ml) or recombinant IL-1RA (0.5, 1.0 and 2.0 µg/ml). Cells were harvested on day 6 and stained with anti-CD3 and 7AAD. After gating on CD3+7AAD- the number of CFSE diluting cells were enumerated using counting beads. Experiments were carried out in duplicates. n=4.</p

Raw data for Figure 3C: Mesoangioblasts and HIDEMs do not interfer with T cell activation

<p>CFSE labelled PBMCs (5 x 104/well) were stimulated with anti CD3/CD28 beads (1 x 104/well) (P+B) in the presence or absence of HIDEMs/mesoangioblasts at HIDEM/mesoangioblast:PBMC = 1:4 ratio. Cells were harvested on day 3, 4, 5 or 6 and analysed for CFSE dilution and expression of CD25 and CD69. The number of CD3+7AAD- cells expressing CD25 or CD69 using counting beads and the % of CD25+ and CD69+ cells were calculated from the data. Experiments were carried out in duplicates. n=2.</p

Raw data for Figure 3B: Mesoangioblasts and HIDEMs suppress T cell proliferation in a dose dependent manner

CFSE labelled PBMCs (5 x 104/well) were stimulated with anti CD3/CD28 beads (1 x 104/well) (P+B) in the presence or absence of HIDEMs/mesoangioblasts at decreasing ratios (HIDEM/mesoangioblast:PBMC). On day 6 cells were harvested and stained with anti-CD3 antibody and 7AAD, and analysed by flow cytometry. CFSE dilution was analysed on gated CD3+ 7AAD- cells. The percentage of CD3+CFSE dividing cells was calculated for each group and compared to the positive control (P+B), followed by plotting against HIDEM/mesoangioblast:PBMC ratios. Experiments were carried out in duplicates. n=