Age-related changes in afferent pathways and urothelial function in the male mouse bladder

Key points •The prevalence of bladder conditions such as overactive bladder syndrome and urinary incontinence significantly increases with age, but how bladder function is altered by ageing is unclear. •Sensory nerves together with the epithelial lining of the bladder known as the urothelium play a key role in mediating bladder function. •In aged male mice we find a significant increase in natural bladder voiding, augmented afferent nerve firing during bladder filling and a significant increase in urothelial responses to purinergic receptor stimulation. •This suggests that with ageing there is increased purinergic transmission in the mouse bladder which may lead to increased sensation and result in bladder hypersensitivity. •These findings help us better understand how the function of the bladder may be affected by advancing age. Abstract The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea

A subpopulation of itch-sensing neurons marked by Ret and somatostatin expression

Itch, the unpleasant sensation that elicits a desire to scratch, is mediated by specific subtypes of cutaneous sensory neuron. Here, we identify a subpopulation of itch-sensing neurons based on their expression of the receptor tyrosine kinase Ret. We apply flow cytometry to isolate Ret-positive neurons from dorsal root ganglia and detected a distinct population marked by low levels of Ret and absence of isolectin B4 binding. We determine the transcriptional profile of these neurons and demonstrate that they express neuropeptides such as somatostatin (Sst), the NGF receptor TrkA, and multiple transcripts associated with itch. We validate the selective expression of Sst using an Sst-Cre driver line and ablated these neurons by generating mice in which the diphtheria toxin receptor is conditionally expressed from the sensory neuron-specific Avil locus. Sst-Cre::AviliDTR mice display normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by interleukin-31 (IL-31) or agonist at the 5HT1F receptor is significantly reduced. Our data provide a molecular signature for a subpopulation of neurons activated by multiple pruritogens. Synopsis This study shows that a subset of DRG neurons expressing the tyrosine kinase Ret and somatostatin function as itch receptor and mediate 5HT1f receptor agonist-induced scratching in mice. Flow cytometric analysis of peripheral sensory neurons is used to isolate multiple Ret-positive subsets. Transcriptional profiling of sensory neurons with low levels of Ret and an absence of IB4 binding reveals co-expression of somatostatin (Sst), interleukin-31 (IL-31) and serotonin receptor 5HT1f. Ablation of Sst-positive neurons reduces scratching responses to IL-31 and 5HT1f agonists in vivo. This study shows that a subset of DRG neurons expressing the tyrosine kinase Ret and somatostatin function as itch receptor and mediate 5HT1f receptor agonist-induced scratching in mice

Ageing and gastrointestinal sensory function: Altered colonic mechanosensory and chemosensory function in the aged mouse

Ageing has a profound effect upon gastrointestinal function through mechanisms that are poorly understood. Here we investigated the effect of age upon gastrointestinal sensory signalling pathways in order to address the mechanisms underlying these changes. In vitro mouse colonic and jejunal preparations with attached splanchnic and mesenteric nerves were used to study mechanosensory and chemosensory afferent function in 3-, 12- and 24-month-old C57BL/6 animals. Quantitative RT-PCR was used to investigate mRNA expression in colonic tissue and dorsal root ganglion (DRG) cells isolated from 3- and 24-month animals, and immunohistochemistry was used to quantify the number of 5-HT-expressing enterochromaffin (EC) cells. Colonic and jejunal afferent mechanosensory function was attenuated with age and these effects appeared earlier in the colon compared to the jejunum. Colonic age-related loss of mechanosensory function was more pronounced in high-threshold afferents compared to low-threshold afferents. Chemosensory function was attenuated in the 24-month colon, affecting TRPV1 and serotonergic signalling pathways. High-threshold mechanosensory afferent fibres and small-diameter DRG neurons possessed lower functional TRPV1 receptor responses, which occurred without a change in TRPV1 mRNA expression. Serotonergic signalling was attenuated at 24 months, but TPH1 and TPH2 mRNA expression was elevated in colonic tissue. In conclusion, we saw an age-associated decrease in afferent mechanosensitivity in the mouse colon affecting HT units. These units have the capacity to sensitise in response to injurious events, and their loss in ageing may predispose the elderly to lower awareness of GI injury or disease

Regolazione epigenetica della Trombomodulina e del Tumor Necrosis Factor mediata da PARP-1 nel mesotelioma maligno

Il cancro esercita un profondo impatto sul sistema emostatico dell’organismo, portando ad uno stato di ipercoagulabilità, che può condurre a complicanze trombotiche. Tra i tumori solidi, il mesotelioma maligno (tumore associato all’esposizione all’amianto) è tra quelli maggiormente interessati da complicanze trombotiche. La trombo-patogenesi è scatenata dal rilascio, da parte delle cellule tumorali, di fattori pro-coagulanti ed infiammatori, che contribuiscono al mantenimento dello stato di ipercoagulabilità. Il Tumor Necrosis Factor (TNF-α) è la citochina che più influisce sul sistema emostatico, mediante una down regolazione delle funzioni anti-coagulanti, quali l’inibizione della Trombomodulina (TM), un anti-coagulante naturale coinvolto in vari processi patologici, inclusi trombosi, infiammazione e cancro. Una ridotta o assente espressione di TM è associata con una prognosi drammatica del tumore. In questo studio, abbiamo analizzato l’espressione di TM e del TNF-α e abbiamo osservato una ridotta espressione di entrambi i geni in campioni di biopsie con mesotelioma maligno (MM) rispetto ai tessuti pleurici sani di controllo (NM). Successivamente, abbiamo indagato il ruolo dell’epigenetica nel silenziamento di questi due geni, con maggior attenzione sull’analisi dei profili di metilazione dei rispettivi promotori, che sono risultati entrambi ipermetilati nei campioni bioptici MM, ma non in quelli sani NM. Il coinvolgimento della metilazione nel silenziamento di TM e TNF-α è stato confermato anche in vitro: le linee cellulari mesoteliali immortalizzate (Met5A) e quelle di mesotelioma maligno (H28) sono state trattate con l’agente demetilante 5’-Aza deossicitidina (5’aza dC) e con l’inibitore delle deacetilasi Tricostatna (TSA). In seguito al trattamento con 5’aza dC, sia TM sia TNF-α erano drasticamente ripristinati nelle cellule H28 ma non nelle Met5A (dove i trattamenti non avevano alcun effetto). Solo per il TNF-α abbiamo osservato una significativa riespressione anche in seguito al trattamento combinato 5’aza dC + TSA nelle cellule H28, facendo ipotizzare un possibile coinvolgimento anche dell’acetilazione nel silenziamento del TNF-α. Infine, abbiamo analizzato il possibile ruolo della Poli(ADP)-ribosio polimerasi (PARP-1) come regolatore epigenetico di questi due geni, valutando gli effetti del suo silenziamento sull’espressione di TM e TNF-α nelle linee cellulari Met5A e H28. Nelle Met5A, il silenziamento di PARP-1 determinava una forte riduzione di TM e di TNF-α, corrispondente ad un profilo di ipermetilazione del loro promotore; al contrario, nelle H28, il silenziamento di PARP-1 determinava una significativa riespressione di TM e una forte riduzione di TNF-α, corrispondente ad un profilo di ipometilazione e di ipermetilazione, rispettivamente. Da questo studio è emerso un chiaro ruolo della metilazione nella regolazione dell’espressione di TM nel mesotelioma maligno e questo meccanismo sembrerebbe essere mediato da PARP-1, che esercita un effetto contrario sull’espressione di TM, nelle cellule di controllo rispetto a quelle tumorali. Più controverso è il meccanismo di regolazione epigenetica del TNF-α, in quanto la metilazione potrebbe non essere il solo meccanismo epigenetico coinvolto; si dovrebbe indagare anche il possibile ruolo dell’acetilazione e l’eventuale coinvolgimento di PARP-1 nell’espressione di questo gene, nel mesotelioma maligno

Alpha-Tocopheryl Succinate Inhibits Autophagic Survival of Prostate Cancer Cells Induced by Vitamin K3 and Ascorbate to Trigger Cell Death

BACKGROUND: The redox-silent vitamin E analog α-tocopheryl succinate (α-TOS) was found to synergistically cooperate with vitamin K3 (VK3) plus ascorbic acid (AA) in the induction of cancer cell-selective apoptosis via a caspase-independent pathway. Here we investigated the molecular mechanism(s) underlying cell death induced in prostate cancer cells by α-TOS, VK3 and AA, and the potential use of targeted drug combination in the treatment of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: The generation of ROS, cellular response to oxidative stress, and autophagy were investigated in PC3 prostate cancer cells by using drugs at sub-toxic doses. We evaluated whether PARP1-mediated apoptosis-inducing factor (AIF) release plays a role in apoptosis induced by the combination of the agents. Next, the effect of the combination of α-TOS, VK3 and AA on tumor growth was examined in nude mice. VK3 plus AA induced early ROS formation associated with induction of autophagy in response to oxidative stress, which was reduced by α-TOS, preventing the formation of autophagosomes. α-TOS induced mitochondrial destabilization leading to the release of AIF. Translocation of AIF from mitochondria to the nucleus, a result of the combinatorial treatment, was mediated by PARP1 activation. The inhibition of AIF as well as of PARP1 efficiently attenuated apoptosis triggered by the drug combination. Using a mouse model of prostate cancer, the combination of α-TOS, VK3 and AA was more efficient in tumor suppression than when the drugs were given separately, without deleterious side effects. CONCLUSIONS/SIGNIFICANCE: α-TOS, a mitochondria-targeting apoptotic agent, switches at sub-apoptotic doses from autophagy-dependent survival of cancer cells to their demise by promoting the induction of apoptosis. Given the grim prognosis for cancer patients, this finding is of potential clinical relevance

Alpha-tocopheryl succinate inhibits autophagic survival of prostate cancer cells induced by vitamin K3 and ascorbate to trigger cell death.

BACKGROUND: The redox-silent vitamin E analog α-tocopheryl succinate (α-TOS) was found to synergistically cooperate with vitamin K3 (VK3) plus ascorbic acid (AA) in the induction of cancer cell-selective apoptosis via a caspase-independent pathway. Here we investigated the molecular mechanism(s) underlying cell death induced in prostate cancer cells by α-TOS, VK3 and AA, and the potential use of targeted drug combination in the treatment of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: The generation of ROS, cellular response to oxidative stress, and autophagy were investigated in PC3 prostate cancer cells by using drugs at sub-toxic doses. We evaluated whether PARP1-mediated apoptosis-inducing factor (AIF) release plays a role in apoptosis induced by the combination of the agents. Next, the effect of the combination of α-TOS, VK3 and AA on tumor growth was examined in nude mice. VK3 plus AA induced early ROS formation associated with induction of autophagy in response to oxidative stress, which was reduced by α-TOS, preventing the formation of autophagosomes. α-TOS induced mitochondrial destabilization leading to the release of AIF. Translocation of AIF from mitochondria to the nucleus, a result of the combinatorial treatment, was mediated by PARP1 activation. The inhibition of AIF as well as of PARP1 efficiently attenuated apoptosis triggered by the drug combination. Using a mouse model of prostate cancer, the combination of α-TOS, VK3 and AA was more efficient in tumor suppression than when the drugs were given separately, without deleterious side effects. CONCLUSIONS/SIGNIFICANCE: α-TOS, a mitochondria-targeting apoptotic agent, switches at sub-apoptotic doses from autophagy-dependent survival of cancer cells to their demise by promoting the induction of apoptosis. Given the grim prognosis for cancer patients, this finding is of potential clinical relevance

Wood dust exposure induces cell transformation through EGFR-mediated OGG1 inhibition

A high risk of neoplastic transformation of nasal and paranasal sinuses mucosa is related to the occupational exposure to wood dust. However, the role of occupational exposures in the aetiology of the airway cancers remains largely unknown. Here, an in vitro model was performed to investigate the carcinogenic effect of wood dusts. Human bronchial epithelial cells were incubated with hard and soft wood dusts and the DNA damage and response to DNA damage evaluated. Wood dust exposure induced accumulation of oxidised DNA bases, which was associated with a delay in DNA repair activity. By exposing cells to wood dust at a prolonged time, wood dust-initiated cells were obtained. Initiated-cells were able to form colonies in soft agar, and to induce blood vessel formation. These cells showed extensive autophagy, reduced DNA repair, which was associated with reduced OGG1 expression and oxidised DNA base accumulation. These events were found related to the activation of EGFR/AKT/mTOR pathway, through phosphorylation and subsequent inactivation of tuberin. The persistence in the tissue of wood dusts, their repetitious binding with EGFR may continually trigger the activation switch, leading to chronic down-regulation of genes involved in DNA repair, leading to cell transformation and proliferation

Vector Aided Microenvironment programming (VAMP): reprogramming the TME with MVA virus expressing IL-12 for effective antitumor activity

Background Tumor microenvironment (TME) represents a critical hurdle in cancer immunotherapy, given its ability to suppress antitumor immunity. Several efforts are made to overcome this hostile TME with the development of new therapeutic strategies modifying TME to boost antitumor immunity. Among these, cytokine-based approaches have been pursued for their known immunomodulatory effects on different cell populations within the TME. IL-12 is a potent pro-inflammatory cytokine that demonstrates striking immune activation and tumor control but causes severe adverse effects when systemically administered. Thus, local administration is considered a potential strategy to achieve high cytokine concentrations at the tumor site while sparing systemic adverse effects.Methods Modified Vaccinia Ankara (MVA) vector is a potent inducer of pro-inflammatory response. Here, we cloned IL-12 into the genome of MVA for intratumoral immunotherapy, combining the immunomodulatory properties of both the vector and the cargo. The antitumor activity of MVA-IL-12 and its effect on TME reprogramming were investigated in preclinical tumor models. RNA sequencing (RNA-Seq) analysis was performed to assess changes in the TME in treated and distal tumors and the effect on the intratumoral T-cell receptor repertoire.Results Intratumoral injection of MVA-IL-12 resulted in strong antitumor activity with the complete remission of established tumors in multiple murine models, including those resistant to checkpoint inhibitors. The therapeutic activity of MVA-IL-12 was associated with very low levels of circulating cytokine. Effective TME reprogramming was demonstrated on treatment, with the reduction of immunosuppressive M2 macrophages while increasing pro-inflammatory M1, and recruitment of dendritic cells. TME switch from immunosuppressive into immunostimulatory environment allowed for CD8 T cells priming and expansion leading to tumor attack.Conclusions Intratumoral administration of MVA-IL-12 turns immunologically ‘cold’ tumors ‘hot’ and overcomes resistance to programmed cell death protein-1 blockade