Cipriani, Gianluca

Age-related gastrointestinal dysfunction is common, but the mechanisms of aging-associated smooth muscle failure remain unclear. We show that aging in mice slows whole gut and colonic transit, increases regional stiffness, and reduces smooth muscle contractility. Inducible smooth muscle cell (SMC)– specific deletion of Piezo1 preserved youthful transit and force generation, whereas Piezo1 activation in young mice phenocopied aging-associated transit delay. Single-cell transcriptomics, RNA velocity, stiffness-controlled cell and tissue cultures, and pharmacologic studies revealed that Piezo1 couples increased stiffness to Ca 2+ –calcineurin–NFAT signaling, loss of contractile gene programs, leading to age- related contractile loss and contractile-to-synthetic SMC remodeling and gut wall stiffening. Human intestinal SMCs supported conservation of this pathway, and PIEZO1 gain-of-function carriers showed a trend toward delayed colonic transit. Thus, maladaptive SMC Piezo1 mechanotransduction is a targetable mechanism of aging-associated gut dysmotility. Aging alters organ structure, yet how these changes drive functional decline remains poorly defined. We show that progressive stiffening of the gut with age activates the mechanosensitive ion channel Piezo1 in smooth muscle cells, initiating a maladaptive response that impairs contractility and slows intestinal transit. On the other hand, deletion of smooth muscle Piezo1 prevents age-related gut dysmotility. These findings reveal that age-related physical shifts actively drive functional decline through mechanotransduction pathways. By identifying Piezo1 as a critical link between tissue mechanics and organ aging, this work provides a framework for understanding how mechanical cues contribute to age-related dysfunction in the gut and possibly other smooth muscle organs.

ABSTRACT The enteric nervous system (ENS) is a complex peripheral neural network that coordinates gastrointestinal motility through highly organized synaptic communication. Although tissue-resident muscularis macrophages (MMs) closely associate with enteric neurons, whether they regulate enteric synaptic organization remains unknown. In the central nervous system (CNS), microglia sculpt neural circuits through complement-dependent synaptic remodeling, raising the possibility that analogous neuroimmune mechanisms operate in the gut. Here, we identify a previously unrecognized role for C1qa□ MMs in regulating enteric synaptic homeostasis and gastrointestinal motility. Using macrophage-specific constitutive and inducible C1qa deletion models, single-cell RNA sequencing, enteric synaptosome proteomics, physiology, and advanced imaging, we demonstrate that loss of MMs-derived C1qa increases enteric synaptic density without altering neuronal numbers. C1qa deficiency induced broad transcriptional changes in enteric neurons and macrophages, including altered synapse-associated, lysosomal, and endocytic programs. Proteomic analysis revealed that enteric synapses share a conserved molecular architecture with brain synapses while exhibiting distinct gastrointestinal-specific complement-associated synaptic networks enriched for structural and receptor-localization pathways. Functionally, macrophage-specific C1qa deletion altered excitatory and inhibitory enteric neurotransmission, enhanced cholinergic signaling, reduced nitrergic responses, and accelerated gastrointestinal transit, while smooth muscle responsiveness remained preserved. C1qa□ MMs displayed transcriptional and functional features consistent with a phagocytic synapse-remodeling phenotype, including enrichment of complement, lysosomal, and engulfment pathways. Loss of C1qa impaired macrophage phagocytic activity both in vitro and in vivo and was associated with synapse accumulation and altered macrophage morphology. Importantly, inducible deletion of C1qa in adulthood recapitulated the synaptic and motility phenotypes, demonstrating that C1qa□ MMs continuously regulate enteric synaptic organization beyond development. Together, these findings identify a complement-dependent neuroimmune mechanism that regulates enteric circuit organization and gut motility, establishing MMs as active modulators of adult ENS synaptic homeostasis. Abstract Figure

Abstract Interstitial cells of Cajal (ICC) regulate gastrointestinal motility by generating electrical slow waves that orchestrate downstream calcium (Ca 2 + ) waves and coordinate smooth muscle contractions. A reduced ICC number has been reported in diabetic gastroparesis patients and in mouse models of delayed gastric emptying (DGE). However, functional changes in ICC propagating across the gastric regions in intact stomach are not well understood. The aim of this study was to develop an imaging approach to visualize ICC Ca 2+ waves in mouse stomach and determine spatiotemporal changes in mice with DGE. Tamoxifen‐treated B6.GCaMP5‐KitCreERT2 female mice were used as non‐diabetic controls (Non‐Db), while mice treated with streptozocin to induce diabetes were categorized as having normal (Db‐NGE) or delayed (Db‐DGE) gastric emptying based on emptying of solids. The ICC Ca 2+ network was imaged from excised intact mouse stomachs and subsequently opened along the lesser curvature and pinned flat. ICC Ca 2+ waves typically propagated along the greater curvature from the fundus–corpus line toward the antrum. The propagation length of ICC Ca 2+ activity was significantly decreased in Db‐DGE compared to Non‐Db and Db‐NGE and correlated to longer GE times. There was also a decrease in frequency, and the velocity was significantly faster in stomachs from Db‐DGE compared to Non‐Db and Db‐NGE. All experimental groups exhibited similar proportions of aberrant Ca 2+ propagation patterns (e.g., retrograde waves), but ectopic initiation only occurred in Db‐DGE stomachs. Thus, DGE is possibly driven by dysfunction in Ca 2+ activity in the ICC network that propagates rapidly over short distances, resulting in uncoordinated, non‐phasic muscle activity. image Key points Dysfunction of the interstitial cells of Cajal (ICC) has been implicated in diabetic gastroparesis but the changes in spatiotemporal properties of ICC network activity are not known. We developed an ex vivo Ca 2+ imaging approach to visualize ICC network activity across the entire stomach and determined changes in ICC Ca 2+ activity in a diabetic mouse model of delayed gastric emptying (GE). Decreased frequency and increased velocity of ICC Ca 2+ activity was observed in stomachs of diabetic mice with delayed GE. Ectopic initiation was a key feature associated with delayed GE. The propagation length of ICC Ca 2+ activity was inversely correlated to GE times and significantly reduced in stomach preparations from diabetic mice with delayed GE.

Background: Diabetic gastroparesis (DG) is associated with a loss in anti-inflammatory muscularis macrophages (MMs), contributing to delayed gastric emptying (DGE). Lyve1 macrophages, known for their anti-inflammatory phenotype, are involved in lipid metabolism across organs. While diabetes is typically associated with increased lipid levels, the dependence of lipid handling on altered function of Lyve1+ MMs in the stomach remains unknown. Aim: To investigate lipid accumulation in MMs and test the effect of palmitate and CD36 on Lyve1+ macrophages. Methods: FACS sorted CD45+ cells were isolated from the gastric muscularis of non-diabetic mice and analyzed using 10X Genomics Cellranger and Seurat package. Gene Set Enrichment Analysis was performed to identify enriched pathways. Separately, the cells underwent flow cytometry to identify Lyve1+/- MMs then determining lipid accumulation by LipidTox intensity. RAW 264.7 macrophage cells were cultured with macrophage colony stimulating factor (M-CSF), IL-4, and dexamethasone for 7 days to induce Lyve1 expression. RNA was collected from cells treated with palmitate and a CD36 inhibitor, Sulfosuccinimidyl oleate (SSO), for RT-qPCR. Lyve1+ and Lyve1- macrophages treated with 300µM palmitate overnight were collected for lipidomic analysis. Untargeted lipidomic analysis was done with Thermo Orbitrap Exploris 120, and data were analyzed using principal component analysis for group clustering and unpaired t-tests for differential analysis. Results: Unbiased clustering analysis revealed 6 transcriptionally distinct gastric MM populations, one of which was notably enriched in Lyve1 expression. Pathway analysis identified that Lyve1 enriched cluster was associated with lysosomal pathways and receptors involved in lipid uptake, such as CD36, as well as anti-inflammatory markers such as HO1 and CD206. Flow cytometry quantification showed that Lyve1+ MMs had higher accumulation of lipids compared to Lyve1- MMs (10.1±0.7 vs 2.9±0.2 mean fluorescence intensity/cell count, p=0.0002). RT-qPCR validated that Lyve1+ RAW 264.7 macrophages exhibited significantly higher expression of Lyve1 (6.95 times fold change, p=0.023) and CD36 (3.27 times fold change, p=0.011) compared to Lyve1- macrophages. Post palmitate treatment, Lyve1+ macrophages had increased expression of the pro-apoptotic gene caspase-3 (12.15-times fold change, p=0.033), as well as the fatty acid oxidation marker ACADM (4.98 times fold change, p=0.045). Notably, these differences were not found in the presence of SSO. Lipidomic analysis identified 171 differentially expressed lipids, particularly increased levels of ceramides and diacylglycerols in Lyve1+ macrophages compared to Lyve1- macrophages when both are treated with palmitate. Conclusions: Lyve1+ macrophages preferentially uptake lipids compared to Lyve1- macrophages consistent with increased expression of CD36. Lipid treatment of Lyve1+ macrophages increases expression of apoptosis and oxidation markers in a CD36-dependent manner that may lead to cell loss. These mechanisms may contribute to Lyve1+ MMs loss in DG. Funding: DK127992; DK129297; DK115255; ANMS Young Investigator grant; AGA grant#36; P30DK084567; Robert and Arlene Kogod Center on Aging Fundamental Mechanisms of Aging Award. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Objective Macrophages are highly plastic immune cells that maintain tissue homeostasis through their roles in immune surveillance, repair, and remodeling. Emerging evidence reveals that macrophages, residing in tissues with widely varying stiffness, are profoundly influenced by the mechanical properties of their microenvironment. Methods Relevant publications on macrophage mechanoregulation published up to January 2026 were identified through systematic searches of PubMed, EMBASE, and Web of Science using combinations of keywords related to macrophages, mechanosensing, mechanotransduction, and mechanical stimuli (e.g., stiffness, stretch, and shear stress). Articles were screened based on relevance to macrophage biology and mechanistic insights. Results Mechanical cues are sensed through mechanosensors such as Piezo1, TRPV4, and integrins, which integrate these signals to regulate immune surveillance, inflammation, tissue repair, and remodeling. Dysregulation of these pathways contributes to the pathogenesis of multiple diseases, including fibrosis, atherosclerosis, and neurodegeneration. Conclusion Macrophage mechanobiology represents a critical regulatory axis in tissue homeostasis and disease. Targeting mechanosensing pathways offers promising therapeutic opportunities to modulate inflammation and enhance tissue regeneration.

Using 32 immune cell surface markers, we identified 23 monocyte clusters in murine blood. Diabetic gastroparesis was associated with a significant decrease in two circulating classical monocyte-like clusters and an upregulation of the Ccr2-Ccl2 axis in the gastric muscularis propria, suggesting increased tissue monocyte migration. This study offers new targets by pointing to a possible role for two classical monocyte subsets connected to the Ccr2- Ccl2 axis.

Background: Gastrointestinal (GI) disorders like chronic constipation are more prevalent with aging. Aging GI tract becomes stiffer and exhibits delayed GI transit. Smooth muscle cells (SMCs) are involved in controlling GI transit and show reduced contractility with age. Piezo1 is a mechanically activated ion channel that responds to membrane tension by opening its pore to allow flow of ions such as calcium (Ca2+) and regulates various cellular processes through Ca2+-calcineurin signaling pathway. We wondered GI stiffness changes might control SMC contractile to non-contractile phenotype through Piezo1. Objective: To determine how substrate stiffness regulates GI SMC contractile phenotype through Piezo1. Hypothesis: SMC Piezo1 senses GI tissue stiffness changes and alters SMC phenotype. Methods: Pathway analysis on human colon single cell sequencing data was performed using Gene Set Enrichment Analysis (GSEA) pre-ranked module with publicly available gene-sets from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Immortalized human jejunal SMCs (HuSMCs) and freshly dissociated SMCs from control (CTRL, Myh11-creERT2) and SMC specific Piezo1 knockout [Myh11-creERT2xPiezo1 Flox (Piezo1-KO)] mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrates to model age-dependent shifts in intestinal stiffness for 48 hours either in the presence/absence of Piezo1 siRNA, Piezo1 agonist (Yoda1, 1μM), and calcineurin blocker (Cyclosporin A, CsA, 1μM) and used for Ca2+ imaging, RT-qPCR and immunostaining. Results: GSEA showed Piezo1+ve SMCs were significantly enriched in pathways involved in the regulation of Ca2+ signaling compared to Piezo1−ve SMCs (p<0.05). HuSMCs and primary SMCs showed increased Piezo1 expression (HuSMCs:1.1±0.2-fold, n=8, p<0.05; primary SMCs:2.1±0.1-fold, n=3, p<0.05) and cytoplasmic Ca2+ (3.6-fold, n=10, p<0.05) in response to Yoda1 on stiff substrates compared to soft. HuSMCs on stiff substrate showed decreased expression of contractile SMC markers smoothelin ( SMTN:0.7±0.2-fold, n=7, p<0.05) and transgelin ( TAGLN:0.6±0.1-fold, n=7, p<0.05) which was rescued by Piezo1 siRNA treatment ( SMTN:1.2±0.1-fold, n=2-7, p<0.05; TAGLN:1.15±0.02-fold, n=2-7, p<0.05) (Piezo1 is necessary). Stimulation by Yoda1 of HuSMCs on the soft substrate resulted in decreased expression of SMTN (0.7±0.3-fold, n=2-7, p<0.05) and TAGLN (0.7±0.1-fold, n=2-7, p<0.05) (Piezo1 is suffcient). CTRL primary SMCs on stiff substrate showed decreased expression of Smtn (0.4±0.1-fold, n=3, p<0.05) and Tagln (0.3±0.2-fold, n=3, p<0.05) compared to SMCs on soft substrate which was reversed when SMCs from Piezo1-KO mice were cultured on these substrates ( Smtn:1.8±0.5-fold, n=3, p<0.05; Tagln:2.4±0.07-fold, n=3, p<0.05). Treatment of CsA in HuSMCs cultured on stiff substrate also rescued from loss of SMTN (1.3±0.1-fold, n=3, p<0.05) and TAGLN (1.7±0.3-fold, n=3, p<0.05) expression and prevented nuclear factor of activated T cells (NFAT) nuclear translocation. Summary and Conclusion: GI SMC Piezo1 senses stiffness changes and regulates SMC phenotype through Ca2+-calcineurin signaling pathway. Targeting Piezo1 might be protective from age induced reduced GI SMC contractility. NIH DK052766, DK123549. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AbstractBackgroundGastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase‐1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin‐10 (IL‐10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL‐10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno‐associated viruses (AAVs) in diabetic mice with delayed GE.MethodsC57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9‐cre, scAAV9‐GFP, or scAAV9‐HO1 particles. Changes to GE were assessed weekly utilizing our [13C]‐octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression.Key ResultsDelayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9‐cre and in 4/5 mice that received the scAAV9‐GFP, whereas mice that received scAAV9‐HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4–6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9‐GFP‐treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE.Conclusions and InferencesInjection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

AbstractBackground and AimMuscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine.MethodsThe distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy.Key ResultsImmunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα‐positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell‐to‐cell contacts with smooth muscle cells and TCs/PDGFRα‐positive cells but not with ICCs and enteric neurons.Conclusion & InferencesThis study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell‐to‐cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα‐positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

Abstract Background Gastric emptying is a complex physiological process regulating the division of a meal into smaller partitions for the small intestine. Disrupted gastric emptying contributes to digestive disease, yet current measures may not reflect different mechanisms by which the process can be altered. Methods We have developed high temporal resolution solid and liquid gastric emptying breath tests in mice using [ 13 C]‐octanoic acid and off axis‐ integrated cavity output spectroscopy ( OA ‐ ICOS ). Stretched gamma variate and 2‐component stretched gamma variate models fit measured breath excretion data. Key Results These assays detect acceleration and delay using pharmacological (7.5 mg/kg atropine) or physiological (nutrients, cold exposure stress, diabetes) manipulations and remain stable over time. High temporal resolution resolved complex excretion curves with 2 components, which was more prevalent in mice with delayed gastric emptying following streptozotocin‐induced diabetes. There were differences in the gastric emptying of Balb/c vs C57Bl6 mice, with slower gastric emptying and a greater occurrence of two‐phase gastric emptying curves in the latter strain. Gastric emptying of C57Bl6 could be accelerated by halving the meal size, but with no effect on the occurrence of two‐phase gastric emptying curves. A greater proportion of two‐phase gastric emptying was induced in Balb/c mice with the administration of PYY (8‐80 nmol) 60 min following meal ingestion. Conclusions and Inferences Collectively, these results demonstrate the utility of high temporal resolution gastric emptying assays. Two‐phase gastric emptying is more prevalent than previously reported, likely involves intestinal feedback, but contributes little to the overall rate of gastric emptying.