BACKGROUND: The extracellular vesicles (EVs) secreted by adipose tissue-derived stromal cells (ASC) are microenvironment modulators in tissue regeneration by releasing their molecular cargo, including miRNAs. However, the influence of ASC-derived extracellular vesicles (ASC-EVs) on endothelial cells (ECs) and vascularisation is poorly understood. The present study aimed to determine the pro-angiogenic effects of ASC-EVs and explore their miRNA profile.
METHODS: EVs were isolated from normoxic and hypoxic cultured ASC conditioned culture medium. The miRNA expression profile was determined by miRseq, and EV markers were determined by Western blot and immunofluorescence staining. The uptake dynamics of fluorescently labelled EVs were monitored for 24 h. ASC-EVs\' pro-angiogenic effect was assessed by sprouting ex vivo rat aorta rings in left ventricular-decellularized extracellular matrix (LV dECM) hydrogel or basement membrane hydrogel (Geltrex®).
RESULTS: ASC-EVs augmented vascular network formation by aorta rings. The vascular network topology and stability were influenced in a hydrogel scaffold-dependent fashion. The ASC-EVs were enriched for several miRNA families/clusters, including Let-7 and miR-23/27/24. The miRNA-1290 was the highest enriched non-clustered miRNA, accounting for almost 20% of all reads in hypoxia EVs.
CONCLUSIONS: Our study revealed that ASC-EVs augment in vitro and ex vivo vascularisation, likely due to the enriched pro-angiogenic miRNAs in EVs, particularly miR-1290. Our results show promise for regenerative and revascularisation therapies based on ASC-EV-loaded ECM hydrogels.

BACKGROUND: In reconstructive surgery, local flaps might develop tissue necrosis or partial flap loss especially after previous irradiation, which may be necessary in many tumor entities. The application of stem cells seems promising to improve flap perfusion and might be a possible solution to optimize flap survival.
METHODS: Twenty rats received harvesting of bilateral random pattern fasciocutaneous flaps. The right flaps received 20 Gy ionizing radiation 4 weeks prior to the surgery, while the left flaps served as the non-irradiated control. After flap harvest, four different stem cell mixtures (5 × 106 ASC, ASC-HUVEC, MSC, MSC-HUVEC) were applied under both right and left flaps using 1 mL fibrin glue as the delivery vehicle. Flap size and its necrotic area were examined clinically. Two weeks after the surgery, HE staining and immunohistochemical staining for CD68 and ERG, as well as PCR analysis (Interleukin 6, HIF-1α and VEGF), were performed.
RESULTS: Application of ASCs, ASCs-HUVECs and MSCs resulted in a lower number of CD68-stained cells compared to the no cell group. The expression of Hif1α was higher in the ASC group compared to those in the MSC and previously treated no cell groups. Treatment with MSCs and MSCs-HUVECs prevented shrinking of the flaps in this series.
CONCLUSIONS: Application of ASCs, MSCs and ASCs-HUVECs was shown to have an antiinflammatory effect. Treatment with MSCs and MSCs-HUVECs can prevent early shrinking of the flaps.

(1) Background: Peptides are appealing as pharmacological materials because they are easily produced, safe, and tolerable. Despite increasing gum-care awareness, periodontitis is still prevalent and is influenced by factors like high sugar consumption, smoking, and aging. Porphyromonas gingivalis is considered a major etiologic agent of periodontitis and activates the NLR family pyrin domain containing 3 (NLRP3) but is absent in melanoma 2 (AIM2) inflammasomes, resulting in pro-inflammatory cytokine release. (2) Methods: We examined the anti-inflammatory effects of 18 peptides derived from human stromal cell-derived factor-1 (SDF-1) on THP-1 macrophages. Inflammation was induced by P. gingivalis, and the anti-inflammatory effects were analyzed using molecular biological techniques. In a mouse periodontitis model, alveolar bone resorption was assessed using micro-CT. (3) Results: Of the 18 SDF-1-derived peptides, S10 notably reduced IL-1β and TNF-α secretion. S10 also diminished the P. gingivalis-induced expression of NLRP3, AIM2, ASC (apoptosis-associated speck-like protein), caspase-1, and IL-1β. Furthermore, S10 attenuated the enhanced TLR (toll-like receptor) signaling pathway and decreased the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). In addition, S10 mitigated alveolar bone loss in our P. gingivalis-induced mouse model of periodontitis. (4) Conclusions: S10 suppressed TLR/NF-κB/NLRP3 inflammasome signaling and the AIM2 inflammasome in our P. gingivalis-induced murine periodontitis model, which suggests that it has potential use as a therapeutic treatment for periodontitis.

Epilepsy is one of the most prevalent and serious brain disorders and affects over 70 million people globally. Antiseizure medications (ASMs) relieve symptoms and prevent the occurrence of future seizures in epileptic patients but have a limited effect on epileptogenesis. Addressing the multifaceted nature of epileptogenesis and its association with the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation requires a comprehensive understanding of the underlying mechanisms of these medications for the development of targeted therapeutic strategies beyond conventional antiseizure treatments. Several types of NLRP3 inhibitors have been developed and their effect has been validated both in in vitro and in vivo models of epileptogenesis. In this review, we discuss the advances in understanding the regulatory mechanisms of NLRP3 activation as well as progress made, and challenges faced in the development of NLRP3 inhibitors for the treatment of epilepsy.

NLRs constitute a large, highly conserved family of cytosolic pattern recognition receptors that are central to health and disease, making them key therapeutic targets. NLRC5 is an enigmatic NLR with mutations associated with inflammatory and infectious diseases, but little is known about its function as an innate immune sensor and cell death regulator. Therefore, we screened for NLRC5\'s role in response to infections, PAMPs, DAMPs, and cytokines. We identified that NLRC5 acts as an innate immune sensor to drive inflammatory cell death, PANoptosis, in response to specific ligands, including PAMP/heme and heme/cytokine combinations. NLRC5 interacted with NLRP12 and PANoptosome components to form a cell death complex, suggesting an NLR network forms similar to those in plants. Mechanistically, TLR signaling and NAD+ levels regulated NLRC5 expression and ROS production to control cell death. Furthermore, NLRC5-deficient mice were protected in hemolytic and inflammatory models, suggesting that NLRC5 could be a potential therapeutic target.

Background: Myelodysplastic syndromes (MDS) are determined by ineffective hematopoiesis and bone marrow cytological dysplasia with somatic gene mutations and chromosomal abnormalities. Accumulating evidence has revealed the pivotal role of NLRP3 inflammasome activation and pyroptotic cell death in the pathogenesis of MDS. Although MDS can be diagnosed with a variety of morphologic and cytogenetic tests, most of these tests have limitations or problems in practice. Materials and Methods: In the present study, we evaluated the expression of genes that form the inflammasome (NLRP3, ASC, and CASP1) in bone marrow specimens of MDS patients and compared the results with those of other leukemias to evaluate their diagnostic value for MDS. Primary samples of this observational cohort study were collected from aspiration samples of patients with myelodysplastic syndromes (27 cases) and patients with non-myelodysplastic syndrome hematological cancers (45 cases). After RNA extraction and c.DNA synthesis, candidate transcripts and housekeeping transcripts were measured by real-time PCR method (SYBER Green assay). Using Kruskal-Wallis the relative gene expressions were compared and differences with p value less than 0.05 were considered as significant. Discrimination capability, cut-off, and area under curve (AUC) of all markers were analyzed with recessive operation curve (ROC) analysis. Results: We found that Caspase-1 and ASC genes expressed at more levels in MDS specimens compared to non-MDS hematological malignancies. A relative average expression of 10.22 with a p-value of 0.001 and 1.86 with p=0.019 was detected for Caspase-1 and ASC, respectively. ROC curve analysis shows an AUC of 0.739 with p=0.0001 for Caspase-1 and an AUC of 0.665 with p=0.0139 for ASC to MDS discrimination. Conclusion: Our results show that Caspase-1 and ASC gene expression levels can be used as potential biomarkers for MDS diagnosis. Prospective studies with large sample numbers are suggested.

Chronic venous disease (CVD) comprises a spectrum of morphofunctional disorders affecting the venous system, affecting approximately 1 in 3 women during gestation. Emerging evidence highlights diverse maternofetal implications stemming from CVD, particularly impacting the placenta. While systemic inflammation has been associated with pregnancy-related CVD, preliminary findings suggest a potential link between this condition and exacerbated inflammation in the placental tissue. Inflammasomes are major orchestrators of immune responses and inflammation in different organs and systems. Notwithstanding the relevance of inflammasomes, specifically the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3)- which has been demonstrated in the placentas of women with different obstetric complications, the precise involvement of this component in the placentas of women with CVD remains to be explored. This study employs immunohistochemistry and real-time PCR (RT-qPCR) to examine the gene and protein expression of key components in both canonical and non-canonical pathways of the NLRP3 inflammasome (NLRP3, ASC-apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain-caspase 1, caspase 5, caspase 8, and interleukin 1β) within the placental tissue of women affected by CVD. Our findings reveal a substantial upregulation of these components in CVD-affected placentas, indicating a potential pathophysiological role of the NLRP3 inflammasome in the development of this condition. Subsequent investigations should focus on assessing translational interventions addressing this dysregulation in affected patient populations.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a crucial complication of long-term chronic diabetes that can lead to myocardial hypertrophy, myocardial fibrosis, and heart failure. There is increasing evidence that DCM is associated with pyroptosis, a form of inflammation-related programmed cell death. Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor β superfamily, which regulates oxidative stress, inflammation, and cell survival to mitigate myocardial hypertrophy, myocardial infarction, and vascular injury. However, the role of GDF11 in regulating pyroptosis in DCM remains to be elucidated. This research aims to investigate the role of GDF11 in regulating pyroptosis in DCM and the related mechanism.
RESULTS: Mice were injected with streptozotocin (STZ) to induce a diabetes model. H9c2 cardiomyocytes were cultured in high glucose (50 mM) to establish an in vitro model of diabetes. C57BL/6J mice were preinjected with adeno-associated virus 9 (AAV9) intravenously via the tail vein to specifically overexpress myocardial GDF11. GDF11 attenuated pyroptosis in H9c2 cardiomyocytes after high-glucose treatment. In diabetic mice, GDF11 alleviated cardiomyocyte pyroptosis, reduced myocardial fibrosis, and improved cardiac function. Mechanistically, GDF11 inhibited pyroptosis by preventing inflammasome activation. GDF11 achieved this by specifically binding to apoptosis-associated speck-like protein containing a CARD (ASC) and preventing the assembly and activation of the inflammasome. Additionally, the expression of GDF11 during pyroptosis was regulated by peroxisome proliferator-activated receptor α (PPARα).
CONCLUSIONS: These findings demonstrate that GDF11 can treat diabetic cardiomyopathy by alleviating pyroptosis and reveal the role of the PPARα-GDF11-ASC pathway in DCM, providing ideas for new strategies for cardioprotection.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation.
METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR.
RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2.
CONCLUSIONS: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Autism is four times more prevalent in males than females. To study whether this reflects a difference in genetic predisposition attributed to autosomal rare variants, we evaluated the sex differences in effect size of damaging protein-truncating and missense variants on autism predisposition in 47,061 autistic individuals, then compared effect sizes between individuals with and without cognitive impairment or motor delay. Although these variants mediated differential likelihood of autism with versus without motor or cognitive impairment, their effect sizes on the liability scale did not differ significantly by sex exome-wide or in genes sex-differentially expressed in the cortex. Although de novo mutations were enriched in genes with male-biased expression in the fetal cortex, the liability they conferred did not differ significantly from other genes with similar loss-of-function intolerance and sex-averaged cortical expression. In summary, autosomal rare coding variants confer similar liability for autism in females and males.