Activity Of Interstitial Cells Research Articles

Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).

Mechanism of miR-221-3p inhibiting cadmium-induced apoptosis of TM3 cells through DDIT4

To investigate the mechanism of DNA-damage-inducible transcript 4(DDIT4)targeting miR-221-3p in microRNA(miRNA) on cadmium-induced apoptosis of mouse testicular stromal cells. The activity of mouse testicular interstitial cells(TM3) was detected by CCK-8 after exposure to different concentrations of cadmium(0, 10, 20, 30, 40 μmol/L). Total RNA was extracted from cadmium-treated TM3 cells, and the significantly differentially expressed miRNA was screened with fold change(FC)>1.2 and P<0.05 as the criterion. TM3 cells were divided into blank control group, negative control group, cadmium exposure group(CdCl_2, 20 μmol/L), and cadmium+miR-221-3p mimic group. miR-221-3p mimic group was transfected into TM3 cells first, combined with cadmium exposure for 24 hours. The cell morphology was detected by Hoechst staining, and the apoptosis rate was analyzed by flow cytometry. Quantitative real-time PCR(qRT-PCR) and Western blot were used to detect DDIT4 expression. Dual luciferase reporter gene assay verified the binding of miR-221-3p to DDIT4. The function of DDIT4 and its relationship with apoptosis were analyzed by bioinformatics. The expression levels of B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein(BAX) were observed after overexpression of miR-221-3p. Cadmium treatment of TM3 cells could reduce cell activity and there was a dose-effect relationship. The cell morphology showed that compared with the control group, the cells were wrinkled and the nuclei were heavily stained, and the apoptosis rate increased to 19.66%±0.45%(P<0.01). Compared with the cadmium exposure group, the normal morphologic cells increased in the cadmium exposure +miR-221-3p mimic group, and the apoptosis rate decreased to 13.76%±0.37%(P<0.05). The expression level of miR-221-3p was down-regulated(P<0.01), and the expression level of DDIT4 was up-regulated(P<0.05). Bioinformatics analysis and dual luciferase report analysis showed that DDIT4 was one of the target genes of miR-221-3p. Compared with the cadmium exposure group, the expression level of DDIT4 in the cadmium+miR-221-3p mimic group was down-regulated(P<0.05), and the ratio of Bcl-2/BAX was increased from 0.54±0.03 to 0.71±0.04. miR-221-3p inhibits cadmium-induced apoptosis of TM3 cells by targeting DDIT4.

5-Hydroxytryptamine Enhances the Pacemaker Activity of Interstitial Cells of Cajal in Mouse Colon.

We examined the localization of the 5-hydroxytryptamine (5-HT) receptor and its effects on mouse colonic interstitial cells of Cajal (ICCs) using electrophysiological techniques. Treatment with 5-HT increased the pacemaker activity in colonic ICCs with depolarization of membrane potentials in a dose-dependent manner. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers blocked pacemaker activity and 5-HT-induced effects. Moreover, an adenylate cyclase inhibitor inhibited 5-HT-induced effects, and cell-permeable 8-bromo-cAMP increased the pacemaker activity. Various agonists of the 5-HT receptor subtype were working in colonic ICCs, including the 5-HT4 receptor. In small intestinal ICCs, 5-HT depolarized the membrane potentials transiently. Adenylate cyclase inhibitors or HCN blockers did not show any influence on 5-HT-induced effects. Anoctamin-1 (ANO1) or T-type Ca2+ channel blockers inhibited the pacemaker activity of colonic ICCs and blocked 5-HT-induced effects. A tyrosine protein kinase inhibitor inhibited pacemaker activity in colonic ICCs under controlled conditions but did not show any influence on 5-HT-induced effects. Among mitogen-activated protein kinase (MAPK) inhibitors, a p38 MAPK inhibitor inhibited 5-HT-induced effects on colonic ICCs. Thus, 5-HT's effect on pacemaker activity in small intestinal and colonic ICCs has excitatory but variable patterns. ANO1, T-type Ca2+, and HCN channels are involved in 5-HT-induced effects, and MAPKs are involved in 5-HT effects in colonic ICCs.

Nanomedicines for an Enhanced Immunogenic Cell Death-Based In Situ Cancer Vaccination Response.

Cancer vaccines have shown tremendous potential in preventing and treating cancer by providing immunogenic antigens to initiate specific tumor immune responses. An in situ vaccine prepared from an autologous tumor can mobilize a patient's own tumor cell lysate as a reservoir of specific antigens, thus triggering a broad immune response and diverse antitumor immunity in an individually tailored manner. Its efficacy is much better than that of conventional vaccines with a limited number of epitopes. Several conventional therapies, including radiotherapy (RT), chemotherapeutics, photodynamic therapy (PDT), and photothermal therapy (PTT) can activate an anticancer in situ vaccine response by inducing immunogenic cell death (ICD), triggering the exposure of tumor-associated antigens (TAAs), cancerous testis antigens, neoantigens, and danger-associated molecular patterns (DAMPs) with low cost. However, the immunogenicity of dying tumor cells is low, making released antigens and DAMPs insufficient to initiate a robust immune response against malignant cancer. Moreover, the immunosuppressive tumor microenvironment (TME) severely hinders the infiltration and sensitization of effector immune cells, causing tolerogenic immunological effects.Herein, we mainly focus on the research in developing nanoplatforms to surmount the major challenges met by ICD-based in situ vaccines. We first summarized a variety of nanotechnologies that enable enhanced immunogenicity of dying cancer cells by enhancing antigenicity and adjuvanticity. The robust antigenicity was obtained via regulating the tumor cells death mode or the dying state to amplify the recognition of tumor debris by professional antigen-presenting cells (APCs). The adjuvanticity was potentiated by raising the level or intensifying the activity of endogenous adjuvants or promoting the intelligent delivery of exogenous immunostimulants to activate immune cell recruitment and promote antigen presentation. Additionally, versatile approaches to reverse immunosuppressive TME to boost the in situ tumor vaccination response are also highlighted in detail. On one hand, by modulating the cell metabolism in TME, the expansion and activity of effector versus immunosuppressive cells can be optimized to improve the efficiency of in situ vaccines. On the other hand, regulating cellular components in TME, such as reversing adverse immune cell phenotypes or inhibiting the activity of interstitial cells, can also significantly enhance the ICD-based antitumor immunotherapy effect. Finally, our viewpoint on the future challenges and opportunities in this hopeful area is presented. We expect that this Account can offer much more insight into the design, planning, and development of cutting-edge in situ tumor vaccine platforms, promoting more attention and academic-industry collaborations, accelerating the advanced progress of in situ tumor vaccine-based immunotherapy in the clinic.

Abstract P1039: Soluble Extracellular Matrix Protein Induces Senescence In Aortic Valve Interstitial Cells To Up-regulate Valvular Osteogenic Activity

Background: Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in the elderly, and elevated osteogenic activity of aortic valve interstitial cells (AVICs) play a critical role in CAVD progression to severe aortic valve stenosis. While soluble extracellular matrix (ECM) proteins, including biglycan and matrilin 2, have been found to up-regulate the osteogenic activity in human AVICs, the mechanism underlying the cellular and molecular mechanisms remain unclear. As greater densities of senescent AVICs were observed in human calcified aortic valves, we tested the hypothesis that soluble ECM protein induces senescence in AVICs to up-regulate valvular osteogenic activity. Methods and Results: Human AVICs were isolated from normal and CAVD aortic valves. Levels of P16 and P21 were assessed by immunoblotting, and senescent changes is identified using senescence-associated β-galactosidase (SA-β-gal) staining. AVICs from diseased valves displayed higher levels of P16 and P21, greater SA-β-gal staining and active calcium deposition. Soluble matrilin 2 (2.0 μg/ml) up-regulated the expression of P16 and P21, and enhanced SA-β-gal staining in normal AVICs, leading to greater calcium deposition. Further, knockdown of P16 markedly reduced calcium deposition in AVICs exposed to soluble matrilin 2. The impact of soluble matrlin 2 is associated with activation of the M6PR-ERK1/2 pathway. Blocking or knockdown of M6PR (M6P, 10 mM or siRNA, 1.0 nM), or inhibition of ERK1/2 (PD98059, 25 μM) attenuated the effect of soluble matrilin 2 on AVIC senescence and resultant up-regulation of osteogenic activity. Conclusion: AVICs from aortic valves of patients with CAVD have higher levels of senescence activity. Soluble matrilin 2, via the M6PR-ERK1/2 pathway, induces AVIC senescence to promote the osteogenic activity. Targeting the D-M6PR-ERK1/2 pathway may suppress AVIC senescence and attenuate aortic valve calcification associated with CAVD progression.

Network Pharmacological Analysis and Experimental Validation of the Effect of Smilacis Glabrae Rhixoma on Gastrointestinal Motility Disorder.

Gastrointestinal motility disorder (GMD) is a disease that causes digestive problems due to inhibition of the movement of the gastrointestinal tract and is one of the diseases that reduce the quality of life of modern people. Smilacis Glabrae Rhixoma (SGR) is a traditional herbal medicine for many diseases and is sometimes prescribed to improve digestion. As a network pharmacological approach, we searched the TCMSP database for SGR, reviewed its constituents and target genes, and analyzed its relevance to gastrointestinal motility disorder. The effects of the SGR extract on the pacemaker activity in interstitial cells of Cajal (ICC) and gastric emptying were investigated. In addition, using the GMD mouse model through acetic acid (AA), we investigated the locomotor effect of SGR on the intestinal transit rate (ITR). As a result of network pharmacology analysis, 56 compounds out of 74 candidate compounds of SGR have targets, the number of targets is 390 targets, and there are 904 combinations. Seventeen compounds of SGR were related to GMD, and as a result of comparing the related genes with the GMD-related genes, 17 genes (active only) corresponded to both. When looking at the relationship network between GMD and SGR, it was confirmed that quercetin, resveratrol, SCN5A, TNF, and FOS were most closely related to GMD. In addition, the SGR extract regulated the pacemaker activity in ICC and recovered the delayed gastric emptying. As a result of feeding the SGR extract to AA-induced GMD mice, it was confirmed that the ITR decreased by AA was restored by the SGR extract. Through network pharmacology, it was confirmed that quercetin, resveratrol, SCN5A, TNF, and FOS were related to GMD in SGR, and these were closely related to intestinal motility. Based on these results, it is suggested that SGR in GMD restores digestion through the recovery of intestinal motility.

Effect of adenosine-1 receptor activation on pacemaker activity of interstitial cells of Cajal from mouse colon.

Adenosine plays an important role on gastrointestinal (GI) motility through adenosine receptors. Interstitial cells of Cajal (ICC) are pacemaker cells that regulate GI smooth muscle activity. The functional role and its signal mechanism of adenosine on the pacemaker activity were investigated using whole-cell patch clamp, RT-PCR, and intracellular Ca2+-imaging with ICC from mouse colon. Adenosine depolarized the membrane potentials and increased the pacemaker potential frequency, which was blocked by a selective A1-receptor antagonist, but not A2a-, A2b, or A3-receptor antagonist. A selective A1 receptor agonist represented similar effects as those of adenosine and mRNA transcript of A1-receptor was expressed in ICC. The adenosine-induced effects were blocked by phospholipase C (PLC) and a Ca2+-ATPase inhibitor. Adenosine increased spontaneous intracellular Ca2+ oscillations, as seen fluo4/AM. Both hyperpolarization-activated cyclic nucleotide (HCN) channel inhibitors and adenylate cyclase inhibitors blocked the adenosine-induced effects. And adenosine increased the basal cellular adenylate cyclase activity in colonic ICC. However, adenosine and adenylate cyclase inhibitors did not show any influence on pacemaker activity in small intestinal ICC for a comparison with that of the small intestine. These results suggest adenosine modulates the pacemaker potentials by acting HCN channels- and intracellular Ca2+- dependent mechanisms through A1-receptor. Therefore, adenosine may be a therapeutic target in colonic motility disorders.

Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation.

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT-/- mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor β1 (TGFβ1) and collagen (COL1A1) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

Telocytes and endometriosis.

Endometriosis involving the presence and growth of glands and stroma outside the uterine cavity is a common, inflammatory, benign gynecologic disease. Nevertheless, no single theory can exactly account for the pathogenesis of endometriosis. Telocytes, a kind of novel mesenchymal cells, have been suggested to be crucial in promoting angiogenesis and increasing the activity of endometrial interstitial cells and inflammatory cells. Given above roles, telocytes may be considered as the possible pathogenesis of endometriosis. We reviewed the current literature on telocytes. The following aspects were considered: (A) the telocytes' typical characteristics, function, and morphological changes in endometriosis; (B) the potential role of telocytes in endometriosis by impacting the inflammation, invasion, and angiogenesis; (C) telocytes as the potential treatment options for endometriosis.

Ca2+ Signaling Is the Basis for Pacemaker Activity and Neurotransduction in Interstitial Cells of the GI Tract.

Years ago gastrointestinal motility was thought to be due to interactions between enteric nerves and smooth muscle cells (SMCs) in the tunica muscularis. Thus, regulatory mechanisms controlling motility were either myogenic or neurogenic. Now we know that populations of interstitial cells, c-Kit+ (interstitial cells of Cajal or ICC), and PDGFRα+ cells (formerly "fibroblast-like" cells) are electrically coupled to SMCs, forming the SIP syncytium. Pacemaker and neurotransduction functions are provided by interstitial cells through Ca2+ release from the endoplasmic reticulum (ER) and activation of Ca2+-activated ion channels in the plasma membrane (PM). ICC express Ca2+-activated Cl- channels encoded by Ano1. When activated, Ano1 channels produce inward current and, therefore, depolarizing or excitatory effects in the SIP syncytium. PDGFRα+ cells express Ca2+-activated K+ channels encoded by Kcnn3. These channels generate outward current when activated and hyperpolarizing or membrane-stabilizing effects in the SIP syncytium. Inputs from enteric and sympathetic neurons regulate Ca2+ transients in ICC and PDGFRα+ cells, and currents activated in these cells conduct to SMCs and regulate contractile behaviors. ICC also serve as pacemakers, generating slow waves that are the electrophysiological basis for gastric peristalsis and intestinal segmentation. Pacemaker types of ICC express voltage-dependent Ca2+ conductances that organize Ca2+ transients, and therefore Ano1 channel openings, into clusters that define the amplitude and duration of slow waves. Ca2+ handling mechanisms are at the heart of interstitial cell function, yet little is known about what happens to Ca2+ dynamics in these cells in GI motility disorders.

Post-Cryo Survival of Rat Testicular Interstitial Cells in Hydroxyethyl Starch-Based Media

The development of cryoprotective serum-free or xeno-free media is required for safe use of cryopreserved testicular material for transplantation. In this study, the solutions containing 10% fetal bovine serum (FBS) or 5 mg/ml bovine serum albumin (BSA) did not significantly enhance the general survival of interstitial cells (ICs) after cryopreservation but increase their metabolic activity and steroid producing cell (HSD+-cells) survival. The use of 50 and 100 mg/ml hydroxyethyl starch (HES) in DMSO-based cryoprotective solutions instead of BSA or FBS enabled the improvement of the IC general survival and the survival of HSD+-cells. The use of HES supplemented media allowed to decrease the dimethyl sulfoxide (DMSO) concentration from 1.4 to 0.7 M and to preserve the amount and metabolic activity of ICs. Thus, designing cryoprotective media containing DMSO and HES can facilitate the formulation of serum-free solutions for cryopreservation that in turn paves a way for implementation of the use of cryopreserved material for practical medicine.

The impact of cryopreservation with polyvinyl alcohol on the survival and functional activity of rat testis interstitial cells

The impact of cryopreservation with polyvinyl alcohol on the survival and functional activity of rat testis interstitial cells

Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

Inhibition of PP2A enhances the osteogenic differentiation of human aortic valvular interstitial cells via ERK and p38 MAPK pathways

AimsTo investigate the role of PP2A in calcified aortic valve disease (CAVD). Materials and methodsThe expressions of PP2A subunits were detected by real-time polymerase chain reaction (RT-PCR) and western blot in aortic valves from patients with CAVD and normal controls, the activities of PP2A were analyzed by commercial assay kit at the same time. Aortic valve calcification of mice was evaluated through histological and echocardiographic analysis. ApoE−/− mice and ApoE−/− mice injected intraperitoneally with PP2A inhibitor LB100 were fed a high-cholesterol diet for 24 weeks. Immunofluorescent staining was used to locate the cell-type in which PP2A activity was decreased, the PP2A activity of valvular interstitial cells (VICs) treated with osteogenic induction medium was assessed by western blot and commercial assay kit. After changing the activity of VICs through pharmacologic and genetic intervention, the osteoblast differentiation and mineralization were assessed by western blot and Alizarin Red staining. Finally, the mechanism was clarified by using several specific inhibitors. Key findingsPP2A activity was decreased both in calcified aortic valves and human VICs under osteogenic induction. The PP2A inhibitor LB100 aggravated the aortic valve calcification of mice. Furthermore, PPP2CA overexpression inhibited osteogenic differentiation of VICs, whereas PPP2CA knockdown promoted the process. Further study revealed that the ERK/p38 MAPKs signaling pathways mediated the osteogenic differentiation of VICs induced by PP2A inactivation. SignificanceThis study demonstrated that PP2A plays an important role in CAVD pathophysiology, PP2A activation may provide a novel strategy for the pharmacological treatment of CAVD.

Na+/Ca2 + Exchange and Pacemaker Activity of Interstitial Cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical slow waves in gastrointestinal (GI) smooth muscles. Slow waves organize basic motor patterns, such as peristalsis and segmentation in the GI tract. Slow waves depend upon activation of Ca2+-activated Cl– channels (CaCC) encoded by Ano1. Slow waves consist of an upstroke depolarization and a sustained plateau potential that is the main factor leading to excitation-contraction coupling. The plateau phase can last for seconds in some regions of the GI tract. How elevated Ca2+ is maintained throughout the duration of slow waves, which is necessary for sustained activation of CaCC, is unknown. Modeling has suggested a role for Na+/Ca2+ exchanger (NCX) in regulating CaCC currents in ICC, so we tested this idea on murine intestinal ICC. ICC of small and large intestine express NCX isoforms. NCX3 is closely associated with ANO1 in ICC, as shown by immunoprecipitation and proximity ligation assays (PLA). KB-R7943, an inhibitor of NCX, increased CaCC current in ICC, suggesting that NCX, acting in Ca2+ exit mode, helps to regulate basal [Ca2+]i in these cells. Shifting NCX into Ca2+ entry mode by replacing extracellular Na+ with Li+ increased spontaneous transient inward currents (STICs), due to activation of CaCC. Stepping ICC from −80 to −40 mV activated slow wave currents that were reduced in amplitude and duration by NCX inhibitors, KB-R7943 and SN-6, and enhanced by increasing the NCX driving force. SN-6 reduced the duration of clustered Ca2+ transients that underlie the activation of CaCC and the plateau phase of slow waves. Our results suggest that NCX participates in slow waves as modeling has predicted. Dynamic changes in membrane potential and ionic gradients during slow waves appear to flip the directionality of NCX, facilitating removal of Ca2+ during the inter-slow wave interval and providing Ca2+ for sustained activation of ANO1 during the slow wave plateau phase.

Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells.

Background: Calcific aortic valve disease (CAVD) is a chronic inflammatory disease. Soluble extracellular matrix (ECM) proteins can act as damage-associated molecular patterns and may induce valvular inflammation. Matrilin-2 is an ECM protein and has been found to elevate the pro-osteogenic activity in human aortic valve interstitial cells (AVICs). Klotho, an anti-aging protein, appears to have anti-inflammatory properties. The effect of matrilin-2 and Klotho on AVIC inflammatory responses remains unclear. Methods and Results: Isolated human AVICs were exposed to matrilin-2. Soluble matrilin-2 induced the production of ICAM-1, MCP-1, and IL-6. It also induced protein kinase R (PKR) activation via Toll-like receptor (TLR) 2 and 4. Pretreatment with PKR inhibitors inhibited NF-κB activation and inflammatory mediator production induced by matrilin-2. Further, recombinant Klotho suppressed PKR and NF-κB activation and markedly reduced the production of inflammatory mediators in human AVICs exposed to matrilin-2. Conclusions: This study revealed that soluble matrilin-2 upregulates AVIC inflammatory activity via activation of the TLR-PKR-NF-κB pathway and that Klotho is potent to suppress AVIC inflammatory responses to a soluble ECM protein through inhibiting PKR. These novel findings indicate that soluble matrilin-2 may accelerate the progression of CAVD by inducing valvular inflammation and that Klotho has the potential to suppress valvular inflammation.

Role of the Plasma Membrane Ca2+-ATPase Pump in the Regulation of Rhythm Generation by an Interstitial Cell of Cajal: A Computational Study

Gastrointestinal motility is based on the rhythmic activity of interstitial cells of Cajal (ICCs). The ICC rhythm generation relies upon characteristic Ca2+-handling mechanisms that involve voltage-gated Ca2+ channels, pumps and exchangers located in the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Mutations, overexpression, and genetic knockdowns of the plasma membrane calcium ATPase (PMCA) pumps have been shown to disrupt calcium signaling and to cause disorders in other cell systems. Using an ICC biophysical model, we investigated the effects of PMCA pump upregulation in ICC rhythm generation. We found that, depending on the PMCA maximum pumping rate, the ICC model generates voltage and Ca2+ oscillations with different characteristics or becomes silent. The model predicts the coexistence of activity regimes near a canonical set of the parameters. One of these regimes is a silent regime that would indicate gastric dysmotility.

Lp(A)-Induced Metabolic Activity In Valve Interstitial Cells Drives Inflammation And Ultimately Calcification

Background and Aims: Calcific Aortic Valve Stenosis (CAVS) is the most common form of valvular heart disease and to date, no effective treatments slowing down disease progression are available. Lipoprotein(a) [Lp(a)], a major carrier of oxidized phospholipids (oxPLs), is a causal, genetic risk factor for CAVS. Valve Interstitial Cells (VICs) are crucial in driving calcification and progressive valvular narrowing. To date, the exact molecular mechanism remains unknown. However, previous studies suggest that Lp(a) induces metabolic reprogramming of the vessel wall, thereby driving sustained inflammation and atherogenesis. We hypothesized that the inflammatory and calcific response of Lp(a) is mediated by Lp(a)-induced metabolic alterations of VICs. Targeting these metabolic changes could potentially be used to reverse inflammation and prevent CAVS progression.

Influence of Platelet-Rich and Platelet-Poor Plasma on Endogenous Mechanisms of Skeletal Muscle Repair/Regeneration.

The morpho-functional recovery of injured skeletal muscle still represents an unmet need. None of the therapeutic options so far adopted have proved to be resolutive. A current scientific challenge remains the identification of effective strategies improving the endogenous skeletal muscle regenerative program. Indeed, skeletal muscle tissue possesses an intrinsic remarkable regenerative capacity in response to injury, mainly thanks to the activity of a population of resident muscle progenitors called satellite cells, largely influenced by the dynamic interplay established with different molecular and cellular components of the surrounding niche/microenvironment. Other myogenic non-satellite cells, residing within muscle or recruited via circulation may contribute to post-natal muscle regeneration. Unfortunately, in the case of extended damage the tissue repair may become aberrant, giving rise to a maladaptive fibrotic scar or adipose tissue infiltration, mainly due to dysregulated activity of different muscle interstitial cells. In this context, plasma preparations, including Platelet-Rich Plasma (PRP) and more recently Platelet-Poor Plasma (PPP), have shown advantages and promising therapeutic perspectives. This review focuses on the contribution of these blood-derived products on repair/regeneration of damaged skeletal muscle, paying particular attention to the potential cellular targets and molecular mechanisms through which these products may exert their beneficial effects.

Spontaneous Activity in Urethral Smooth Muscle.

The urethra is a muscular tube that extends from the bladder neck and is composed of an inner layer of smooth muscle referred to as the internal urethral sphincter and an outer layer of striated muscle which forms the external urethral sphincter. The smooth muscle layer can be separated into an inner layer of longitudinally orientated smooth muscle and an outer, relatively thinner, layer of circular muscle. Tonic contraction of both the smooth and striated muscle components of the urethra generates a urethral closure pressure which exceeds intravesical pressure in the bladder to maintain urinary continence. It is likely that contraction of urethral smooth muscle is involved in the long-term maintenance of tone, since it can achieve this at relatively low energy cost, whereas the striated muscle contributes more to the rise in urethral tone that accompanies increases in bladder pressure secondary to coughing or other sudden increases in intra-abdominal pressure. The level of urethral smooth muscle tone is regulated by several autonomic neurotransmitters, including noradrenaline, acetylcholine, ATP and nitric oxide. However, it is also clear that urethral smooth muscle is capable of generating significant tone in the absence of neural input. In this chapter we will discuss the mechanisms responsible for contraction of urethral smooth muscle, with specific focus on the role of ion channels and Ca2+ handling proteins to this process. The mechanisms underlying spontaneous activity in urethral interstitial cells(UICs), putative pacemaker cells of the urethra, will also be examined along with the modulation of these mechanisms by key excitatory and inhibitory neurotransmitters.