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Background: Lipoprotein(a) [Lp(a)]-induced inflammation contributes to coronary artery spasm (CAS) by the contraction of vascular smooth muscle cells. However,the interaction between Lp(a) and soluble CD36 (sCD36)/interleukin (IL)-6/RAS Homolog Family Member A (RhoA)-GTP signaling pathway has not been evaluated. Methods: We investigated the relevance of Lp(a)/CD36 signaling in CAS patient monocyte-derived macrophages (PMDMs) and a human coronary artery smooth muscle cell (HCASMC) line using expression profile correlation analyses,molecular docking,RNA sequencing,flow cytometry,immunoblotting,and quantitative reverse transcription polymerase chain reaction. Results: Plasma Lp(a) and sCD36 levels in 41 CAS patients were significantly higher ( p = 0.001) and positively correlated (r 2 = 0.3145,p < 0.001),a trend not observed in 36 non-CAS controls. RNA sequencing indicated a significant co-overexpression of CD36 and RhoA in Lp(a)-treated CAS PMDMs and HCASMCs,of which the mRNA and protein expression of CD36 and RhoA were significantly enhanced ( p < 0.001) dose-dependently. Lp(a) rather than LDL preferentially induced CD80+ PMDM (M1) polarization. In HCASMCs,the CD36 knockdown using either short hairpin RNA or natural biflavonoid amentoflavone suppressed Lp(a)-upregulated protein expression of CD36,RhoA-GTP,IL-6,tumor necrosis factor (TNF)-α,nuclear factor (NF)-κB,and CD80; however,overexpressed CD36 increased their levels. Lp(a) decreased and amentoflavone increased the epigenetic expression of CD36 inhibitors,miR-335-5p,and miR-448,respectively. Reciprocally,an miRNA inhibitor or mimic could magnify or diminish Lp(a)-induced CD36,TNF-α,NF-κB and IL-6 expressions in HCASMCs,respectively. Conclusions: Elevated Lp(a) levels upregulate the CD36-dependent TNF-α/NF-κB/IL-6/RhoA-GTP signaling pathway in CAS PMDMs and HCASMCs,indicating that Lp(a)/CD36 inflammatory signaling,HCASMC activation,and macrophage M1 polarization mediate CAS development. View Publication

Suspension bath bioprinting,whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing,has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here,we report a systematic investigation of suspension bath properties that support the printing,fusion,and culture of spheroid-based bioinks without added stabilisation. Specifically,agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150 µ m in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however,the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing,fusion,and culture of spheroid-based inks,either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture. View Publication

Tissue remodeling is a key feature of allergic rhinitis (AR),but its underlying molecular mechanisms remain unclear. Lysyl oxidase-like 2 (LOXL2),a regulator of tissue remodeling,has not been studied in AR. Proteomic analysis was performed on nasal mucosal tissues from 8 AR patients and 8 healthy controls (HCs) to identify differentially expressed proteins (DEPs). The top three upregulated DEPs and their association with tissue remodeling markers were validated by immunofluorescence,Western blot,and RT-qPCR in an independent cohort of 30 AR patients and 30 HCs. In vitro,human nasal epithelial cells (HNECs) were treated with IL-4,and the effects of candidate protein inhibitors on remodeling were assessed. An AR mouse model was used to evaluate the impact of these inhibitors on nasal inflammation and remodeling. Proteomic analysis revealed a disease-specific protein expression profile in the nasal mucosa of AR patients,with the top three upregulated proteins being LOXL2,TGF-β1,and TIRAP. Tissue validation showed that LOXL2 was significantly upregulated in the nasal mucosa of AR patients compared to HCs and was significantly correlated with EMT markers (TGF-β1,α-SMA,and E-cadherin). In vitro,IL-4 stimulation significantly upregulated LOXL2,TGF-β1,and α-SMA,while downregulating E-cadherin in a dose-dependent manner in human nasal epithelial cells. These effects were reversed by inhibition of LOXL2. Further investigations demonstrated that LOXL2 promotes tissue remodeling through activation of the TGF-β1/Smad signaling pathway. In the AR mouse model,LOXL2 inhibitors significantly reduced nasal mucosal inflammation and tissue remodeling. Our proteomic analysis suggests that LOXL2 may be involved in the pathological remodeling processes of AR,potentially through modulation of the TGF-β1/Smad signaling pathway. These findings provide preliminary evidence that LOXL2 could serve as a candidate biomarker and a possible therapeutic target in AR,warranting further investigation. View Publication

Despite recent approval of monoclonal antibodies that reduce amyloid (Aβ) accumulation,the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimer's disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target,where its weak inhibition activates neuroprotective signalling,benefiting AD mouse models with Aβ and p-Tau pathologies. Rational design and structure‒activity relationship studies yielded mtCI inhibitors profiled in a drug discovery funnel designed to address safety,selectivity,and efficacy. The lead compound C458 is highly protective against Aβ toxicity,has favourable pharmacokinetics,and minimal off-target effects. C458 exhibited excellent brain penetrance,activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted using functional tests,metabolic assessment,in vivo 31 P-NMR spectroscopy,blood cytokine panels,ex vivo electrophysiology,and Western blotting. Chronic oral administration improved long-term potentiation,reduced oxidative stress and inflammation,and enhanced mitochondrial biogenesis,antioxidant signalling,and cellular energetics. Efficacy against Aβ and p-Tau was confirmed in human organoids. These studies provide further evidence that the restoration of mitochondrial function in response to mild energetic stress represents a promising disease-modifying strategy for AD. This research was supported by grants from NIH AG 5549-06,NS1 07265,AG 062135,UG3/UH3 NS 113776,and ADDF 291204 (all to ET); U19 AG069701 (to TK); the Alzheimer’s Association Research Fellowship grant 23AARF-1027342 (to TKON). View Publication

Atezolizumab plus bevacizumab and lenvatinib are standard treatments for unresectable hepatocellular carcinoma; however,tumor markers such as alpha-fetoprotein and des-gamma-carboxy prothrombin have a limited ability to reflect treatment responses. Circulating tumor cells are non-invasive biomarkers associated with cancer stemness and treatment resistance. We assessed circulating tumor cell subsets expressing cancer stem cell markers (CD90,epithelial cell adhesion molecule,CD133,vimentin) using multiparametric flow cytometry at early and maximal response phases in patients receiving atezolizumab plus bevacizumab or lenvatinib. Early decreases in CD90-positive circulating tumor cells after therapy initiation were associated with tumor shrinkage and longer progression-free survival in both groups,as well as prolonged overall survival in the atezolizumab plus bevacizumab group. At maximal response,changes in CD90-positive circulating tumor cells reflected tumor burden more accurately than alpha-fetoprotein or des-gamma-carboxy prothrombin. These findings highlight the potential of CD90-positive circulating tumor cells to become dynamic biomarkers in systemic therapy for unresectable hepatocellular carcinoma. View Publication

Cancer research has long focused on mutations in normal body cells,but this approach has not produced major breakthroughs for most cancers. Our study explores a different concept that some aggressive cancers may actually arise from early reproductive cells called primordial germ cells,which normally develop into eggs and sperm. We created a new experimental model showing how a virus can transform human primordial germ cell-like cells into virus-positive Merkel cell carcinoma,a rare but deadly skin cancer. This model shows that cancers can emerge through changes in developmental states rather than relying solely on genetic mutations. By linking cancer development to early germ cells,our findings suggest a unifying explanation for both germ cell cancers and body cancers. This new perspective may guide more effective approaches to study,diagnose,and treat cancer by focusing on early human development rather than only DNA mutations and later developmental stages. View Publication

With the approval of the antibody-drug conjugate enfortumab vedotin (EV),NECTIN4 has emerged as a bona fide therapeutic target in urothelial carcinoma (UC). Here,we report the development of a NECTIN4-directed chimeric antigen receptor (CAR) T cell,which exhibits reactivity across cells expressing a range of endogenous NECTIN4,with enhanced activity in high expressors. We demonstrate that the PPARγ pathway,critical for luminal differentiation,transcriptionally controls NECTIN4,and that the PPARγ agonist rosiglitazone primes and augments NECTIN4 expression,thereby increasing sensitivity to NECTIN4-CAR T cell-mediated killing. NECTIN4-CAR T cells have potent anti-tumor activity even against EV resistant cells,which largely retain NECTIN4 expression,including in a post-EV biopsy cohort. Our results elucidate a therapeutically actionable mechanism that UC cells use to control NECTIN4 expression and suggest therapeutic approaches that leverage PPARγ agonists for rational combinations with NECTIN4-targeting agents in UC,as well as future potential treatment options for EV-refractory patients. Subject terms: Bladder cancer,Cancer immunotherapy,Cancer therapeutic resistance,Oncology,Bladder cancer View Publication

Despite the success of antiretroviral therapy in suppressing plasma viremia in people living with human immunodeficiency virus type-1 (HIV-1),persistent viral RNA expression in tissue reservoirs is observed and can contribute to HIV-1-induced immunopathology and comorbidities. Infection of long-lived innate immune cells,such as tissue-resident macrophages and microglia may contribute to persistent viral RNA production and chronic inflammation. We recently reported that de novo cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) in macrophages and microglia leads to MDA5 and MAVS-dependent innate immune sensing and induction of type I IFN responses,demonstrating that HIV icRNA is a pathogen-associated molecular pattern (PAMP). In this report,we show that cytoplasmic expression of HIV-1 icRNA also induces NLRP1 inflammasome activation and IL-1β secretion in macrophages and microglia in an RLR- and endosomal TLR-independent manner. Infection of both macrophages and microglia with either replication-competent or single-cycle HIV-1 induced IL-1β secretion,which was attenuated when cytoplasmic expression of viral icRNA was prevented. While IL-1β secretion was blocked by treatment with caspase-1 inhibitors or knockdown of NLRP1 or caspase-1 expression in HIV-infected macrophages,overexpression of NLRP1 significantly enhanced IL-1β secretion in an HIV-icRNA-dependent manner. Immunoprecipitation analysis revealed interaction of HIV-1 icRNA,but not multiply-spliced HIV-1 RNA,with NLRP1,suggesting that HIV-1 icRNA sensing by NLRP1 is sufficient to trigger inflammasome activation. Together,these findings reveal a pathway of NLRP1 inflammasome activation induced by de novo expressed HIV icRNA in HIV-infected myeloid cells. View Publication

Metformin rejuvenates adult rat oligodendrocyte progenitor cells (OPCs) allowing more efficient differentiation into oligodendrocytes and improved remyelination,and therefore is of interest as a therapeutic in demyelinating diseases such as multiple sclerosis (MS). Here,we test whether metformin has a similar effect in human stem cell derived-OPCs. We assess how well human monoculture,organoid and chimera model culture systems simulate in vivo adult human oligodendrocytes,finding most close resemblance in the chimera model. Metformin increases myelin proteins and/or sheaths in all models even when human cells remain fetal-like. In the chimera model,metformin leads to increased mitochondrial area both in the human transplanted cells and in the mouse axons with associated increase of mitochondrial function/metabolism transcripts. Human oligodendrocytes from MS brain donors treated pre-mortem with metformin also express similar transcripts. Metformin’s brain effect is thus not cell-specific,alters metabolism in part through mitochondrial changes and leads to more myelin production. This bodes well for clinical trials testing metformin for neuroprotection. Subject terms: Oligodendrocyte,Multiple sclerosis,Multiple sclerosis,Regeneration and repair in the nervous system View Publication

Epigenetic modulators of the histone deacetylase (HDAC) family control key biological processes and are frequently dysregulated in cancer. There is superior activity of HDAC inhibitors (HDACi) in patients with myeloproliferative neoplasms (MPNs) that carry the Janus kinase-2 point mutant JAK2 V617F . This constitutively active tyrosine kinase activates signal-transducer-and-activator-of-transcription (STAT) transcription factors to promote cell proliferation and inflammatory processes. We reveal that the inhibition of HDAC1/HDAC2 with the clinically advanced HDACi romidepsin,the experimental HDACi entinostat and MERCK60,and genetic depletion of HDAC1/HDAC2 induce apoptosis and long-term growth arrest of primary and permanent MPN cells in vitro and in vivo. This treatment spares normal hematopoietic stem cells and does not compromise blood cell differentiation. At the molecular level,HDAC1 and HDAC2 control the protein stability of SIAH2 through acetylation. Genetic knockout experiments show that SIAH2 accelerates the proteasomal degradation of JAK2 V617F in conjunction with the E2 ubiquitin-conjugating enzyme UBCH8. SIAH2 binds to the surface-exposed SIAH degron motif VLP1002 in the catalytic domain of JAK2 V617F . At the functional level,SIAH2 knockout MPN cells are significantly less sensitive to HDACi. Global RNA sequencing verifies that JAK-STAT signaling is a prime target of SIAH2. Moreover,HDAC1 is an adverse prognostic factor in patients with acute myeloid leukemia ( n = 150,p = 0.02),being a possible complication of MPNs. These insights reveal a previously unappreciated link between HDAC1/HDAC2 as key molecular targets,the still undefined regulation of cytoplasmic-to-nuclear signaling by HDACs,and how HDACi kill JAK2 V617F -positive cells from MPN patients and mice with JAK2 V617F in vitro and in vivo. Subject terms: Haematological cancer,Oncogenes,Target identification,Haematopoietic stem cells View Publication