Background: Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS).However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear.This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, in vitro and in vivo.Methods: Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress in vitro.Endothelium-specific knockout of DKK1 (DKK1 ECKO /APOE -/-) and endothelium-specific overexpression of DKK1 (DKK1 ECTg ) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation in vivo.RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1.Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms.Results: DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs.However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1 ECKO and DKK1 ECTg mice.RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs.Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter.SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1 ECKO /APOE -/-mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency.Conclusions: Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs.These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.
Abstract Ischemia-reperfusion injury (IRI) can be found in transplantation and other ischemic syndromes in multiple organ systems including kindey, brain, heart and liver, this pathogenesis of IRI is complex and incompletely understood. It has been suggested that reoxygenation after hypoxia is a crucial mechanism mediating organ injury in IRI. Well, dendritic cells (DCs), the critical immune cells linking innate and adaptive immunity, are often exposed to various oxygen tensions under physiological and pathological conditions, such as ischemia-reperfusion. However, the effects of various oxygen tensions on DCs remain unclear. In this study, we showed that hypoxia-differentiated DCs expressed lower levels of MHC-|| molecule, co-stimulatory molecules (CD80, CD86) and pro-inflammatory cytokines (IL-1 β, IL-6, TNF-α and IL-12), but higher levels of immunoregulatory cytokine transforming growth factor-beta (TGF-b) than did normoxia-differentiated DCs. However, reoxygenation of the hypoxia-differentiated DCs dramatically upregulateed the aforementioned molecules leading to a mature phenotype. Significantly, these reoxygenated DCs induced syngeneic or allogeneic naïve CD4+ T cells to differentiate into Th1 effector cells. The generation of CD4+CD25+Foxp3+ regulatory T cells (Treg cells) induced by reoxygenated DCs was however down-regulated. These data indicate that hypoxic microenvironment suppresses the maturation and function of murine DCs. Re-exposure of the hypoxia-differentiated DCs to sutured oxygen however results in complete recovery of their mature phenotype and function and drives them to initiate Th1 cell differentiation, suggesting DCs play a critical role in inflammation in ischemia-reperfusion injury (IRI).
Preclinical mouse models that recapitulate some characteristics of coronavirus disease (COVID-19) will facilitate focused study of pathogenesis and virus-host responses. Human agniotensin-converting enzyme 2 (hACE2) serves as an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect people via binding to envelope spike proteins. Herein we report development and characterization of a rapidly deployable COVID-19 mouse model. C57BL/6J (B6) mice expressing hACE2 in the lung were transduced by oropharyngeal delivery of the recombinant human adenovirus type 5 that expresses hACE2 (Ad5-hACE2). Mice were infected with SARS-CoV-2 at Day 4 after transduction and developed interstitial pneumonia associated with perivascular inflammation, accompanied by significantly higher viral load in lungs at Days 3, 6, and 12 after infection compared with Ad5-empty control group. SARS-CoV-2 was detected in pneumocytes in alveolar septa. Transcriptomic analysis of lungs demonstrated that the infected Ad5-hACE mice had a significant increase in IFN-dependent chemokines
Abstract Renal macrophages (RMs) participate in tissue homeostasis, inflammation and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions and metabolic states of these two RM populations remain unclear. Here we investigate and characterize RMs at different ages by conditionally labeling and ablating RMs populations in several transgenic lines. We find that RMs expand and mature in parallel with renal growth after birth, and are mainly derived from fetal liver monocytes before birth, but self-maintain through adulthood with contribution from peripheral monocytes. Moreover, after the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, with the CX3CR1/CX3CL1 signaling axis being essential for the maintenance and regeneration of both EMRMs and BMRMs. Lastly, we show that EMRMs have a higher capacity for scavenging immune complex, and are more sensitive to immune challenge than BMRMs, with this difference associated with their distinct glycolytic capacities.
Rationale: Kupffer cells (KCs) play a crucial role in liver immune homeostasis through interacting with other immune cells and liver sinusoidal endothelial cells (LSECs).However, how KCs exactly interact with these cells for maintaining the homeostasis still require the further investigation.CXCL10 is a chemokine that has been implicated in chemoattraction of monocytes, T cells, NK cells, and dendritic cells, and promotion of T cell adhesion to endothelial cells.Although CXCL10 is also known to participate in the pathogenesis of hepatic inflammation, the degree to which it is functionally involved in the crosstalk between immune cells and regulation of immune response is still unclear.Methods: To dynamically investigate the function of KCs, we used our recently developed rapid cell ablation model, intermedilysin (ILY)/human CD59 (hCD59)-mediated cell ablation tool, to selectively ablate KC pool under normal condition or concanavalin A (Con A)-induced hepatitis.At certain time points after KCs ablation, we performed flow cytometry to monitor the amount of hepatic infiltrating immune cells.mRNA array was used to detect the change of hepatic cytokines and chemokines levels.Cytokines and chemokines in the serum were further measured by LEGENDplex TM mouse proinflammatory chemokine panel and inflammation panel.Evans blue staining and transmission electron microscopy were used to investigate the interaction between KCs and LSECs in steady condition.CXCL10 neutralizing antibody and CXCL10 deficient mouse were used to study the role of CXCL10 in immune cell migration and pathogenesis of Con A-induced hepatitis.Results: At steady state, elimination of KCs results in a reduction of hepatic infiltrating monocytes, T, B, and NK cells and a list of cytokines and chemokines at transcriptional level.In the meantime, the depletion of KCs resulted in increased sinusoidal vascular permeability.In the pathological condition, the KCs elimination rescues Con A-induced acute hepatitis through suppressing proinflammatory immune responses by down-regulation of hepatitis-associated cytokines/chemokines in serum such as CXCL10, and recruitment of infiltrating immune cells (monocytes, T, B, and NK cells).We further documented that deficiency or blockade of CXCL10 attenuated the development of Con A-induced hepatitis associated with reduction of the infiltrating monocytes, especially inflammatory Ly6C hi monocytes.Conclusions: This study supports the notion that KCs actively interact with immune cells and LSECs for maintaining immune response and liver homeostasis.Our data indicate that the interplay between KCs and infiltrated monocytes via CXCL10 contribute to Con A-induced hepatitis.
There is increased interest in immune-based monoclonal antibody therapies for different malignancies because of their potential specificity and limited toxicity. The activity of some therapeutic monoclonal antibodies is partially dependent on complement-dependent cytolysis (CDC), in which the immune system surveys for invading pathogens, infected cells, and malignant cells and facilitates their destruction. CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that protects cells from CDC. However, in certain tumors, CD59 expression is enhanced, posing a significant obstacle for treatment, by hindering effective monoclonal antibody-induced CDC. In this study, we used non-small lung carcinoma cells to characterize the mechanism of a novel CD59 inhibitor: the 114-amino acid recombinant form of the 4th domain of intermedilysin (rILYd4), a pore forming toxin secreted by Streptococcus intermedius. We compared the rates of internalization of CD59 in the presence of rILYd4 or anti-CD59 antibodies and determined that rILYd4 induces more rapid CD59 uptake at early time points. Most significantly, upon binding to rILYd4, CD59 is internalized and undergoes massive degradation in lysosomes within minutes. The remaining rILYd4·CD59 complexes recycle to the PM and are shed from the cell. In comparison, upon internalization of CD59 via anti-CD59 antibody binding, the antibody·CD59 complex is recycled via early and recycling endosomes, mostly avoiding degradation. Our study supports a novel role for rILYd4 in promoting internalization and rapid degradation of the complement inhibitor CD59, and highlights the potential for improving CDC-based immunotherapy.
When total flavones of Hippophae rhamnoides L (TFH) 100 ug/ml were added to the medium. the spontaneous arrhythmias of the cell clusters disappeared in 14/19 experiments. TFH 100 ug/ml significantly counteracted the positive chronotropic effects of CaC12 and isoproterenol. with the maximal response being markedly decreased in the cultured rat heart cells. TFH 100 ug/ml reduced the level of cAMP in both ischemic and nonischemic myocardium in the isolated perfused rat heart. TFH 100 and 400 ug/ml inhibited adenylate cyclase of the rat ventricular myocardium. These results suggest that the antiarrhythmic effects of TFH are exerted on the heart cells directly. and that the calcium antagonizing action and inhibition of adenylate cyclase with a reduction in the cAMP level may be an important mechanism related to the antiarrhythmic effects.
Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.
Abstract Dissecting and understanding the cancer ecosystem, especially that around the tumor margins, which have strong implications for tumor cell infiltration and invasion, are essential for exploring the mechanisms of tumor metastasis and developing effective new treatments. Using a novel tumor border scanning and digitization model enabled by nanoscale resolution-SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we identified a 500 µm-wide zone centered around the tumor border in patients with liver cancer, referred to as “the invasive zone”. We detected strong immunosuppression, metabolic reprogramming, and severely damaged hepatocytes in this zone. We also identified a subpopulation of damaged hepatocytes with increased expression of serum amyloid A1 and A2 (referred to collectively as SAAs) located close to the border on the paratumor side. Overexpression of CXCL6 in adjacent malignant cells could induce activation of the JAK-STAT3 pathway in nearby hepatocytes, which subsequently caused SAAs’ overexpression in these hepatocytes. Furthermore, overexpression and secretion of SAAs by hepatocytes in the invasive zone could lead to the recruitment of macrophages and M2 polarization, further promoting local immunosuppression, potentially resulting in tumor progression. Clinical association analysis in additional five independent cohorts of patients with primary and secondary liver cancer ( n = 423) showed that patients with overexpression of SAAs in the invasive zone had a worse prognosis. Further in vivo experiments using mouse liver tumor models in situ confirmed that the knockdown of genes encoding SAAs in hepatocytes decreased macrophage accumulation around the tumor border and delayed tumor growth. The identification and characterization of a novel invasive zone in human cancer patients not only add an important layer of understanding regarding the mechanisms of tumor invasion and metastasis, but may also pave the way for developing novel therapeutic strategies for advanced liver cancer and other solid tumors.
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