Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures

open10Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability.openScattolini, Valentina; Luni, Camilla; Zambon, Alessandro; Galvanin, Silvia; Gagliano, Onelia; Ciubotaru, Catalin Dacian; Avogaro, Angelo; Mammano, Fabio; Elvassore, Nicola; Fadini, Gian PaoloScattolini, Valentina; Luni, Camilla; Zambon, Alessandro; Galvanin, Silvia; Gagliano, Onelia; Ciubotaru, CATALIN DACIAN; Avogaro, Angelo; Mammano, Fabio; Elvassore, Nicola; Fadini, GIAN PAOL

Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa

The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31-37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

In vitro and in vivo study of NETosis in diabetic wound healing

People with diabetes have an increased risk of developing several serious health problems. One of the most frequent complication is an impaired wound healing. Neutrophils are recruited early to the wound bed and, upon activation, they undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. This process is called NETosis. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, excessive NETosis can damage tissue. The aim of this study was to examine the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in non-healing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components were elevated in the blood of patients with DFUs. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis, but an impaired inducible NETosis. We have set up a system of intravital microscopy to monitor the presence of neutrophils in a mouse model of ulcer; this imaging technique, in addition to FACS detection of histone citrullination, showed that (1) PAD4 activity was increased by diabetes and (2) NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing and its modulation could be a therapeutic strategy to improve outcome of DFU in diabetic patients.La FID, Federazione Internazionale del Diabete, paragona questa patologia ad una vera e propria epidemia inarrestabile. Una delle gravi complicanze causata da questa malattia sono le ulcere nel piede del diabetico (DFU, Diabetic Foot Ulcer): infatti la guarigione delle ferite è compromessa in molti pazienti diabetici e ciò provoca un aumentato rischio di morbilità e mortalità. I neutrofili sono le principali cellule coinvolte nelle prime fasi della guarigione delle ulcere, in quanto sono la principale componente cellulare reclutata in sede di danno tissutale. I neutrofili hanno un ruolo fondamentale nel sistema immunitario innato e rappresentano la prima difesa contro l’infezione da patogeni. Questa attività antimicrobica è resa possibile anche da un tipo di morte cellulare tipico dei neutrofili al quale è stato dato il nome di NETosi. La NETosi si differenzia da necrosi, apoptosi, necroptosi e autofagia. E’ innescata da PAD4 (Peptidil Arginine Deaminase 4) che è un enzima nucleare Ca2+-dipendente appartenente alla superfamiglia delle amidinotransferasi, coinvolto nella citrullinazione degli istoni. A questa fase seguono la decondensazione della cromatina, la migrazione dell’elastasi all’interno del nucleo e la rottura della membrana nucleare con il conseguente rilascio di DNA sottoforma di filamenti. Questi filamenti, chiamati NETs (Neutrophil Extracellular Traps), sono composti da enzimi e proteine granulari e da materiale nucleare, il cui compito è quello di intrappolare fisicamente i patogeni e le loro componenti, fino alla lisi cellulare. Questo processo è alterato nel diabete e può provocare danni ai tessuti, causando un ritardo nella guarigione delle ferite. Lo scopo di questo progetto è stato quello di studiare gli effetti della NETosi sulla guarigione delle ulcere dei pazienti diabetici, avvalendoci anche dell’uso di un modello sperimentale animale. L’analisi proteomica iniziale ci ha permesso di identificare le proteine coinvolte in questo processo; inoltre, abbiamo osservato che nel sangue dei pazienti diabetici vi è una sovra-espressione di alcuni di questi componenti. I neutrofili isolati da questi pazienti mostrano: 1- un incremento della NETosi spontanea; 2- un’alterata NETosi indotta; 3- un’aumentata attività dell’enzima PAD4. Per studiare meglio questo processo abbiamo messo a punto una tecnica di imaging intravitale nell’ulcera di modello murino, che parallelamente all’analisi citofluorimetrica degli istoni citrullinati, ha messo in risalto il coinvolgimento della NETosi nel processo di guarigione delle ulcere diabetiche. Inoltre l’inibizione di PAD4 nel topo, ha dato evidenze sperimentali di un miglioramento nella guarigione delle ulcere. In futuro questo protocollo sperimentale potrebbe essere utilizzato come strategia terapeutica di modulazione della NETosi per migliorare le DFU nei pazienti diabetici

In vitro and in vivo study of NETosis in diabetic wound healing

People with diabetes have an increased risk of developing several serious health problems. One of the most frequent complication is an impaired wound healing. Neutrophils are recruited early to the wound bed and, upon activation, they undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. This process is called NETosis. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, excessive NETosis can damage tissue. The aim of this study was to examine the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in non-healing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components were elevated in the blood of patients with DFUs. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis, but an impaired inducible NETosis. We have set up a system of intravital microscopy to monitor the presence of neutrophils in a mouse model of ulcer; this imaging technique, in addition to FACS detection of histone citrullination, showed that (1) PAD4 activity was increased by diabetes and (2) NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing and its modulation could be a therapeutic strategy to improve outcome of DFU in diabetic patients.La FID, Federazione Internazionale del Diabete, paragona questa patologia ad una vera e propria epidemia inarrestabile. Una delle gravi complicanze causata da questa malattia sono le ulcere nel piede del diabetico (DFU, Diabetic Foot Ulcer): infatti la guarigione delle ferite è compromessa in molti pazienti diabetici e ciò provoca un aumentato rischio di morbilità e mortalità. I neutrofili sono le principali cellule coinvolte nelle prime fasi della guarigione delle ulcere, in quanto sono la principale componente cellulare reclutata in sede di danno tissutale. I neutrofili hanno un ruolo fondamentale nel sistema immunitario innato e rappresentano la prima difesa contro l’infezione da patogeni. Questa attività antimicrobica è resa possibile anche da un tipo di morte cellulare tipico dei neutrofili al quale è stato dato il nome di NETosi. La NETosi si differenzia da necrosi, apoptosi, necroptosi e autofagia. E’ innescata da PAD4 (Peptidil Arginine Deaminase 4) che è un enzima nucleare Ca2+-dipendente appartenente alla superfamiglia delle amidinotransferasi, coinvolto nella citrullinazione degli istoni. A questa fase seguono la decondensazione della cromatina, la migrazione dell’elastasi all’interno del nucleo e la rottura della membrana nucleare con il conseguente rilascio di DNA sottoforma di filamenti. Questi filamenti, chiamati NETs (Neutrophil Extracellular Traps), sono composti da enzimi e proteine granulari e da materiale nucleare, il cui compito è quello di intrappolare fisicamente i patogeni e le loro componenti, fino alla lisi cellulare. Questo processo è alterato nel diabete e può provocare danni ai tessuti, causando un ritardo nella guarigione delle ferite. Lo scopo di questo progetto è stato quello di studiare gli effetti della NETosi sulla guarigione delle ulcere dei pazienti diabetici, avvalendoci anche dell’uso di un modello sperimentale animale. L’analisi proteomica iniziale ci ha permesso di identificare le proteine coinvolte in questo processo; inoltre, abbiamo osservato che nel sangue dei pazienti diabetici vi è una sovra-espressione di alcuni di questi componenti. I neutrofili isolati da questi pazienti mostrano: 1- un incremento della NETosi spontanea; 2- un’alterata NETosi indotta; 3- un’aumentata attività dell’enzima PAD4. Per studiare meglio questo processo abbiamo messo a punto una tecnica di imaging intravitale nell’ulcera di modello murino, che parallelamente all’analisi citofluorimetrica degli istoni citrullinati, ha messo in risalto il coinvolgimento della NETosi nel processo di guarigione delle ulcere diabetiche. Inoltre l’inibizione di PAD4 nel topo, ha dato evidenze sperimentali di un miglioramento nella guarigione delle ulcere. In futuro questo protocollo sperimentale potrebbe essere utilizzato come strategia terapeutica di modulazione della NETosi per migliorare le DFU nei pazienti diabetici

A perspective on NETosis in diabetes and cardiometabolic disorders

Aims: To review the significance of a new type of neutrophil cell death (NETosis) in diabetes and cardiometabolic diseases. Data synthesis: Diabetes and the metabolic syndrome are characterized by activation of the innate immune system. In this framework, neutrophils are front line defences against infections, but can also turn deleterious if abnormally stimulated. NETosis refers to a type of cell death whereby neutrophils release nuclear material and granule enzymes that together form the NETs (neutrophil extracellular traps). As NETs entrap bacteria, NETosis is instrumental to the clearance of microorganisms, but an exaggerated NETosis response can also lead to tissue damage in several pathological conditions. In diabetes, the finely tuned balance of NETosis required to protect the human body from microorganisms yet avoiding self-damage seems to be lost. In fact, in vitro induction of NETosis and circulating concentrations of NET-associated proteins appear to be enhanced in diabetic patients. Furthermore, NETs contribute to endothelial damage, thrombosis, and ischemia/reperfusion injury, making it a novel player in the pathobiology of cardiovascular disease. Though the cellular events taking place during NETosis have been described and directly visualized, its molecular machinery is still incompletely understood. Protein kinase C (PKC) and NADPH oxidase (NOX) are two important targets to counter NETosis in the setting of diabetes. Conclusions: NETosis appears to be part of an abnormal response to damage in diabetes that, in turn, can promote or aggravate end-organ complications. We suggest that this will be a hot topic of investigation in diabetology in the near futur

Mitochondrial Calcium Uptake Is Instrumental to Alternative Macrophage Polarization and Phagocytic Activity

Macrophages are highly plastic and dynamic cells that exert much of their function through phagocytosis. Phagocytosis depends on a coordinated, finely tuned, and compartmentalized regulation of calcium concentrations. We examined the role of mitochondrial calcium uptake and mitochondrial calcium uniporter (MCU) in macrophage polarization and function. In primary cultures of human monocyte-derived macrophages, calcium uptake in mitochondria was instrumental for alternative (M2) macrophage polarization. Mitochondrial calcium uniporter inhibition with KB-R7943 or MCU knockdown, which prevented mitochondrial calcium uptake, reduced M2 polarization, while not affecting classical (M1) polarization. Challenging macrophages with E. coli fragments induced spikes of mitochondrial calcium concentrations, which were prevented by MCU inhibition or silencing. In addition, mitochondria remodelled in M2 macrophages during phagocytosis, especially close to sites of E. coli internalization. Remarkably, inhibition or knockdown of MCU significantly reduced the phagocytic capacity of M2 macrophages. KB-R7943, which also inhibits the membrane sodium/calcium exchanger and Complex I, reduced mitochondria energization and cellular ATP levels, but such effects were not observed with MCU silencing. Therefore, phagocytosis inhibition by MCU knockdown depended on the impaired mitochondrial calcium buffering rather than changes in mitochondrial and cellular energy status. These data uncover a new role for MCU in alternative macrophage polarization and phagocytic activity

Uncarboxylated Osteocalcin Stimulates 25-Hydroxy Vitamin D Production in Leydig Cell Line Through a GPRC6a-Dependent Pathway

Recent studies disclosed a cross talk between testis and bone. By the action of LH, Leydig cells are able to modulate bone metabolism through testosterone and insulin-like factor 3. Moreover, LH modulates the Leydig expression of CYP2R1, the key enzyme involved in vitamin D (Vit D) 25-hydroxylation. However, pathways regulating CYP2R1 expression have been poorly investigated. The cross talk from the bone to the testis of the vitamin D 25-hydroxylase CYP2R1 involves osteocalcin (OC), which is produced by the osteoblasts and stimulates the production of testosterone by the Leydig cells through its putative receptor GPRC6A, a cation-sensing G-protein-coupled receptor. The aim of this study was to investigate the possible action of OC on CYP2R1 expression and 25-hydroxy Vit D (25-OH Vit D) production in a mouse Leydig cell line (MA-10). After confirmation of the expression of GPRC6A by MA-10, we found that stimulation with either human chorionic gonadotropin or uncarboxylated-OC (ucOC) increases CYP2R1 protein expression in a dose-dependent manner and, in turn, increases the release of 25-OH Vit D in culture medium. This effect was abolished by receptor blockade with, respectively, anti-LH receptor and anti-GPRC6A antibodies. Moreover, both agonists converged to phosphorylation of Erk1/2 by a likely differential action on second messengers. Human chorionic gonadotropin induced slow "tonic" increase of intercellular calcium and accumulation of cAMP, whereas ucOC mainly induced phasic increase of cell calcium. Supporting these findings, we found that serum ucOC positively correlated with 25-OH Vit D levels in 40 overweight male patients and 21 controls. Altogether, our results suggest that OC contributes with LH to 25-OH Vit D production by Leydig cells

Pharmacologic PPAR-\u3b3 Activation Reprograms Bone Marrow Macrophages and Partially Rescues HSPC Mobilization in Human and Murine Diabetes

Mobilization of hematopoietic stem/progenitor cells (HSPCs) from the bone marrow (BM) is impaired in diabetes. Excess oncostatin M (OSM) produced by M1 macrophages in the diabetic BM signals through p66Shc to induce Cxcl12 in stromal cells and retain HSPCs. BM adipocytes are another source of CXCL12 that blunts mobilization. We tested a strategy of pharmacologic macrophage reprogramming to rescue HSPC mobilization. In vitro, PPAR-\u3b3 activation with pioglitazone switched macrophages from M1 to M2, reduced Osm expression, and prevented transcellular induction of Cxcl12 In diabetic mice, pioglitazone treatment downregulated Osm, p66Shc and Cxcl12 in the hematopoietic BM, restored the effects of granulocyte-colony stimulation factor (G-CSF), and partially rescued HSPC mobilization, but it increased BM adipocytes. Osm deletion recapitulated the effects of pioglitazone on adipogenesis, which was p66Shc-independent, and double knockout of Osm and p66Shc completely rescued HSPC mobilization. In the absence of OSM, BM adipocytes produced less CXCL12, being arguably devoid of HSPC-retaining activity, whereas pioglitazone failed to downregulate Cxcl12 in BM adipocytes. In diabetic patients under pioglitazone therapy, HSPC mobilization after G-CSF was partially rescued. In summary, pioglitazone reprogrammed BM macrophages and suppressed OSM signaling, but sustained Cxcl12 expression by BM adipocytes could limit full recovery of HSPC mobilization