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Abstract Metabolic reprogramming in malignant cells is a hallmark of cancer that relies on augmented glycolytic metabolism to support their growth, invasion, and metastasis. However, the impact of global adipose metabolism on tumor growth and the drug development by targeting adipose metabolism remain largely unexplored. Here we show that a therapeutic paradigm of drugs is effective for treating various cancer types by browning adipose tissues. Mirabegron, a clinically available drug for overactive bladders, displays potent anticancer effects in various animal cancer models, including untreatable cancers such as pancreatic ductal adenocarcinoma and hepatocellular carcinoma, via the browning of adipose tissues. Genetic deletion of the uncoupling protein 1, a key thermogenic protein in adipose tissues, ablates the anticancer effect. Similarly, the removal of brown adipose tissue, which is responsible for non-shivering thermogenesis, attenuates the anticancer activity of mirabegron. These findings demonstrate that mirabegron represents a paradigm of anticancer drugs with a distinct mechanism for the effective treatment of multiple cancers.

BackgroundThe obesity epidemic has been on the rise due to changes in living standards and lifestyles. To combat this issue, sleeve gastrectomy (SG) has emerged as a prominent bariatric surgery technique, offering substantial weight reduction. Nevertheless, the mechanisms that underlie SG-related bodyweight loss are not fully understood.MethodsIn this study, we conducted a collection of preoperative and 3-month postoperative serum and fecal samples from patients who underwent laparoscopic SG at the First Affiliated Hospital of Shandong First Medical University (Jinan, China). Here, we took an unbiased approach of multi-omics to investigate the role of SG-altered gut microbiota in anti-obesity of these patients. Non-target metabolome sequencing was performed using the fecal and serum samples.ResultsOur data show that SG markedly increased microbiota diversity and Rikenellaceae, Alistipes, Parabacteroides, Bactreoidales, and Enterobacteraies robustly increased. These compositional changes were positively correlated with lipid metabolites, including sphingolipids, glycerophospholipids, and unsaturated fatty acids. Increases of Rikenellaceae, Alistipes, and Parabacteroide were reversely correlated with body mass index (BMI).ConclusionIn conclusion, our findings provide evidence that SG induces significant alterations in the abundances of Rikenellaceae, Alistipes, Parabacteroides, and Bacteroidales, as well as changes in lipid metabolism-related metabolites. Importantly, these changes were found to be closely linked to the alleviation of obesity. On the basis of these findings, we have identified a number of microbiotas that could be potential targets for treatment of obesity.

Abstract Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

The efficacy of anti‐angiogenic treatment by targeting VEGF/VEGF receptors in metastatic clear cell renal cell carcinoma (ccRCC) varies from patient to patient. Discovering the reasons behind this variability could lead to the identification of relevant therapeutic targets. Thus, we investigated the novel splice variants of VEGF that are less efficiently inhibited by anti‐VEGF/VEGFR targeting than the conventional isoforms. By in silico analysis, we identified a novel splice acceptor in the last intron of the VEGF gene resulting in an insertion of 23 bp in VEGF mRNA. Such an insertion can shift the open‐reading frame in previously described splice variants of VEGF (VEGFXXX), leading to a change in the C‐terminal part of the VEGF protein. Next, we analysed the expression of these alternatively spliced VEGF new isoforms (VEGFXXX/NF) in normal tissues and in RCC cell lines by qPCR and ELISA, and we investigated the role of VEGF222/NF (equivalent to VEGF165) in physiological and pathological angiogenesis. Our in vitro data demonstrated that recombinant VEGF222/NF stimulated endothelial cell proliferation and vascular permeability by activating VEGFR2. In addition, VEGF222/NF overexpression enhanced proliferation and metastatic properties of RCC cells, whereas downregulation of VEGF222/NF resulted in cell death. We also generated an in vivo model of RCC by implanting RCC cells overexpressing VEGF222/NF in mice, which we treated with polyclonal anti‐VEGFXXX/NF antibodies. VEGF222/NF overexpression enhanced tumour formation with aggressive properties and a fully functional vasculature, while treatment with anti‐VEGFXXX/NF antibodies slowed tumour growth by inhibiting tumour cell proliferation and angiogenesis. In a patient cohort from the NCT00943839 clinical trial, we investigated the relationship between plasmatic VEGFXXX/NF levels, resistance to anti‐VEGFR therapy and survival. High plasmatic VEGFXXX/NF levels correlated with shorter survival and lower efficacy of anti‐angiogenic drugs. Our data confirmed the existence of new VEGF isoforms that could serve as novel therapeutic targets in patients with RCC that are resistant to anti‐VEGFR therapy.

Abstract Background Tumors possess incessant growth features, and expansion of their masses demands sufficient oxygen supply by red blood cells (RBCs). In adult mammals, the bone marrow (BM) is the main organ regulating hematopoiesis with dedicated manners. Other than BM, extramedullary hematopoiesis is discovered in various pathophysiological settings. However, whether tumors can contribute to hematopoiesis is completely unknown. Accumulating evidence shows that, in the tumor microenvironment (TME), perivascular localized cells retain progenitor cell properties and can differentiate into other cells. Here, we sought to better understand whether and how perivascular localized pericytes in tumors manipulate hematopoiesis. Methods To test if vascular cells can differentiate into RBCs, genome‐wide expression profiling was performed using mouse‐derived pericytes. Genetic tracing of perivascular localized cells employing NG2‐CreERT2:R26R‐tdTomato mouse strain was used to validate the findings in vivo. Fluorescence‐activated cell sorting (FACS), single‐cell sequencing, and colony formation assays were applied for biological studies. The production of erythroid differentiation‐specific cytokine, erythropoietin (EPO), in TME was checked using quantitative polymerase chain reaction (qPCR), enzyme‐linked immunosorbent assay (ELISA, magnetic‐activated cell sorting and immunohistochemistry. To investigate BM function in tumor erythropoiesis, BM transplantation mouse models were employed. Results Genome‐wide expression profiling showed that in response to platelet‐derived growth factor subunit B (PDGF‐B), neural/glial antigen 2 (NG2)+ perivascular localized cells exhibited hematopoietic stem and progenitor‐like features and underwent differentiation towards the erythroid lineage. PDGF‐B simultaneously targeted cancer‐associated fibroblasts to produce high levels of EPO, a crucial hormone that necessitates erythropoiesis. FACS analysis using genetic tracing of NG2+ cells in tumors defined the perivascular localized cell‐derived subpopulation of hematopoietic cells. Single‐cell sequencing and colony formation assays validated the fact that, upon PDGF‐B stimulation, NG2+ cells isolated from tumors acted as erythroblast progenitor cells, which were distinctive from the canonical BM hematopoietic stem cells. Conclusions Our data provide a new concept of hematopoiesis within tumor tissues and novel mechanistic insights into perivascular localized cell‐derived erythroid cells within TME. Targeting tumor hematopoiesis is a novel therapeutic concept for treating various cancers that may have profound impacts on cancer therapy.

Abstract Background Antiangiogenic tyrosine kinase inhibitors (TKIs) provide one of the few therapeutic options for effective treatment of hepatocellular carcinoma (HCC). However, patients with HCC often develop resistance toward antiangiogenic TKIs, and the underlying mechanisms are not understood. The aim of this study was to determine the mechanisms underlying antiangiogenic TKI resistance in HCC. Methods We used an unbiased proteomic approach to define proteins that were responsible for the resistance to antiangiogenic TKIs in HCC patients. We evaluated the prognosis, therapeutic response, and serum insulin‐like growth factor‐binding protein‐1 (IGFBP‐1) levels of 31 lenvatinib‐treated HCC patients. Based on the array of results, a retrospective clinical study and preclinical experiments using mouse and human hepatoma cells were conducted. Additionally, in vivo genetic and pharmacological gain‐ and loss‐of‐function experiments were performed. Results In the patient cohort, IGFBP‐1 was identified as the signaling molecule with the highest expression that was inversely associated with overall survival. Mechanistically, antiangiogenic TKI treatment markedly elevated tumor IGFBP‐1 levels via the hypoxia‐hypoxia inducible factor signaling. IGFBP‐1 stimulated angiogenesis through activation of the integrin α5β1‐focal adhesion kinase pathway. Consequently, loss of IGFBP‐1 and integrin α5β1 by genetic and pharmacological approaches re‐sensitized HCC to lenvatinib treatment. Conclusions Together, our data shed light on mechanisms underlying acquired resistance of HCC to antiangiogenic TKIs. Antiangiogenic TKIs induced an increase of tumor IGFBP‐1, which promoted angiogenesis through activating the IGFBP‐1‐integrin α5β1 pathway. These data bolster the application of a new therapeutic concept by combining antiangiogenic TKIs with IGFBP‐1 inhibitors.

Abstract Despite the development of advanced technologies for interventional coronary reperfusion after myocardial infarction, a substantial number of patients experience high mortality due to myocardial ischemia‐reperfusion (MI/R) injury. An in‐depth understanding of the mechanisms underlying MI/R injury can provide crucial strategies for mitigating myocardial damage and improving patient survival. Here, it is discovered that the 4‐hydroxy‐2‐nonenal (4‐HNE) accumulates during MI/R, accompanied by high rates of myocardial ferroptosis. The loss‐of‐function of aldehyde dehydrogenase 2 (ALDH2), which dissipates 4‐HNE, aggravates myocardial ferroptosis, whereas the activation of ALDH2 mitigates ferroptosis. Mechanistically, 4‐HNE targets glutathione peroxidase 4 (GPX4) for K48‐linked polyubiquitin‐related degradation, which 4‐HNE‐GPX4 axis commits to myocyte ferroptosis and forms a positive feedback circuit. 4‐HNE blocks the interaction between GPX4 and ovarian tumor (OTU) deubiquitinase 5 (OTUD5) by directly carbonylating their cysteine residues at C93 of GPX4 and C247 of OTUD5, identifying OTUD5 as the novel deubiquitinase for GPX4. Consequently, the elevation of OTUD5 deubiquitinates and stabilizes GPX4 to reverse 4‐HNE‐induced ferroptosis and alleviate MI/R injury. The data unravel the mechanism of 4‐HNE in GPX4‐dependent ferroptosis and identify OTUD5 as a novel therapeutic target for the treatment of MI/R injury.

Abstract The circadian clock in animals and humans plays crucial roles in multiple physiological processes. Disruption of circadian homeostasis causes detrimental effects. Here, it is demonstrated that the disruption of the circadian rhythm by genetic deletion of mouse brain and muscle ARNT‐like 1 (Bmal1) gene, coding for the key clock transcription factor, augments an exacerbated fibrotic phenotype in various tumors. Accretion of cancer‐associated fibroblasts (CAFs), especially the alpha smooth muscle actin positive myoCAFs, accelerates tumor growth rates and metastatic potentials. Mechanistically, deletion of Bmal1 abrogates expression of its transcriptionally targeted plasminogen activator inhibitor‐1 (PAI‐1). Consequently, decreased levels of PAI‐1 in the tumor microenvironment instigate plasmin activation through upregulation of tissue plasminogen activator and urokinase plasminogen activator. The activated plasmin converts latent TGF‐β into its activated form, which potently induces tumor fibrosis and the transition of CAFs into myoCAFs, the latter promoting cancer metastasis. Pharmacological inhibition of the TGF‐β signaling largely ablates the metastatic potentials of colorectal cancer, pancreatic ductal adenocarcinoma, and hepatocellular carcinoma. Together, these data provide novel mechanistic insights into disruption of the circadian clock in tumor growth and metastasis. It is reasonably speculated that normalization of the circadian rhythm in patients provides a novel paradigm for cancer therapy.

Antibacterial protein hydrogels are receiving increasing attention in the aspect of bacteria-infected-wound healing. However, bacterial drug resistance and biofilm infections lead to hard healing of wounds, thus the construction of biological agents that can overcome these issues is essential. Here, a simple and universal method to construct antibiotic-free protein hydrogel with excellent biocompatibility and superior antibacterial activity against drug-resistant bacteria and biofilms was developed. The green industrial microbicide tetrakis (hydroxymethyl) phosphonium sulfate (THPS) as cross-linking agent can be quickly cross-linked with model protein bovine serum albumin (BSA) to form antibacterial hydrogel through simple mixing without any other initiators, subsequently promoting drug-resistance bacteria-infected wound healing. This simple gelatinization strategy allows at least ten different proteins to form hydrogels (e.g. BSA, human serum albumin (HSA), egg albumin, chymotrypsin, trypsin, lysozyme, transferrin, myohemoglobin, hemoglobin, and phycocyanin) under the same conditions, showing prominent universality. Furthermore, drug-resistance bacteria and biofilm could be efficiently destroyed by the representative BSA hydrogel (B-Hydrogel) with antibacterial activity, overcoming biofilm-induced bacterial resistance. The in vivo study demonstrated that the B-Hydrogel as wound dressing can promote reepithelization to accelerate the healing of methicillin-resistant staphylococcus aureus (MRSA)-infected skin wounds without inducing significant side-effect. This readily accessible antibiotic-free protein-based hydrogel not only opens an avenue to provide a facile, feasible and general gelation strategy, but also exhibits promising application in hospital and community MRSA disinfection and treatment.

Abstract PROteolysis TArgeting Chimeras (PROTACs) are an emerging class of promising therapeutic modalities that selectively degrade intracellular proteins of interest by hijacking the ubiquitin‐proteasome system. However, the lack of techniques to efficiently transport these degraders to targeted cells and consequently the potential toxicity of PROTACs limit their clinical applications. Here, a strategy of nanoengineered PROTACs, that is, Nano‐PROTACs, is reported, which improves the bioavailability of PROTACs and maximizes their capacity to therapeutically degrade intracellular oncogenic proteins for tumor therapy. The Nano‐PROTACs are developed by encapsulating PROTACs in glutathione (GSH)‐responsive poly(disulfide amide) polymeric (PDSA) nanoparticles and show that ARV@PDSA Nano‐PROTAC, nanoengineered BRD4 degrader ARV‐771, improves BRD4 protein degradation and decreases the downstream oncogene c‐Myc expression. Benefiting from the GSH‐scavenging ability to amply the c‐Myc‐related ferroptosis and cell cycle arrest, this ARV@PDSA Nano‐PROTACs strategy shows superior anti‐tumor efficacy with a low dose administration and good biocompatibility in vivo. The findings reveal the potential of the Nano‐PROTACs strategy to treat a broad range of diseases by dismantling associated pathogenic proteins.

The corneal endothelium is critical for maintaining corneal clarity by mediating hydration through barrier and pump functions. Progressive loss of corneal endothelial cells during aging has been associated with the development of Fuchs endothelial corneal dystrophy (FECD), one of the main causes of cornea-related vision loss. The mechanisms underlying FECD development remain elusive. Single-cell RNA sequencing of isolated healthy human corneas discovered 4 subpopulations of corneal endothelial cells with distinctive signatures. Unsupervised clustering analysis uncovered nuclear enriched abundant transcript 1 (NEAT1), a long non-coding RNA (lncRNA), as the top expressed gene in the C0-endothelial subpopulation, but markedly downregulated in FECD. Consistent with human corneas, a UVA-induced mouse FECD model validated the loss of NEAT1 expression. Loss of NEAT1 function by an in vivo genetic approach reproduced the exacerbated phenotype of FECD by ablating corneal endothelial cells. Conversely, gain of function by a CRISPR-activated adenoviral delivery system protected corneas from UVA-induced FECD. Our findings provide novel mechanistic insights into the development of FECD, and targeting NEAT1 offers an attractive approach for treating FECD.

Downregulation of MHC class I (MHCI) molecules on tumor cells is recognized as a resistance mechanism of cancer immunotherapy. Given that MHCI molecules are potent regulators of immune responses, we postulated that the expression of MHCI by tumor cells influences systemic immune responses. Accordingly, mice-bearing MHCI-deficient tumor cells showed reduced tumor-associated extramedullary myelopoiesis in the spleen. Depletion of natural killer (NK) cells abrogated these differences, suggesting an integral role of immune-regulatory NK cells during tumor progression. Cytokine-profiling revealed an upregulation of TNF-α by NK cells in tumors and spleen in mice-bearing MHCI expressing tumors, and inhibition of TNF-α enhanced host myelopoiesis in mice receiving adoptive transfer of tumor-experienced NK cells. Our study highlights a critical role of NK cells beyond its identity as a killer lymphocyte and more importantly, the potential host responses to a localized tumor as determined by its MHCI expression.

In this single-arm clinical trial, the authors show that treatment of COVID-19 patients with bevacizumab, an anti-vascular endothelial growth factor drug, can improve PaO2/FiO2 ratios and oxygen-support status. Relative to an external control group, bevacizumab shows clinical efficacy by improving oxygenation.

Dufies et al. find high Plk1 expression levels in aggressive clear cell renal cell carcinoma and discover that Plk1 is transcriptionally upregulated in a manner dependent on HIF-2. They also find that high Plk1 expression is correlated to a poor prognosis and resistance to tyrosine kinase inhibitor against VEGF receptor, suggesting a critical role for hypoxia/HIF-2-induced Plk1 in disease progression.

Background: Bacillus Calmette-Guérin (BCG) immunotherapy is the standard-of-care adjuvant therapy for non-muscle-invasive bladder cancer in patients at considerable risk of disease recurrence. Although its exact mechanism of action is unknown, BCG significantly reduces this risk in responding patients but is mainly associated with toxic side-effects in those facing treatment resistance. Methods that allow the identification of BCG responders are, therefore, urgently needed. Methods: Fluorescently labelled UM-UC-3 cells and dissociated patient tumor samples were used to establish zebrafish tumor xenograft (ZTX) models. Changes in the relative primary tumor size and cell dissemination to the tail were evaluated via fluorescence microscopy at three days post-implantation. The data were compared to the treatment outcomes of the corresponding patients. Toxicity was evaluated based on gross morphological evaluation of the treated zebrafish larvae. Results: BCG-induced toxicity was avoided by removing the water-soluble fraction of the BCG formulation prior to use. BCG treatment via co-injection with the tumor cells resulted in significant and dose-dependent primary tumor size regression. Heat-inactivation of BCG decreased this effect, while intravenous BCG injections were ineffective. ZTX models were successfully established for six of six patients based on TUR-B biopsies. In two of these models, significant tumor regression was observed, which, in both cases, corresponded to the treatment response in the patients. Conclusions: The observed BCG-related anti-tumor effect indicates that ZTX models might predict the BCG response and thereby improve treatment planning. More experiments and clinical studies are needed, however, to elucidate the BCG mechanism and estimate the predictive value.

Human pluripotent stem cells (hPSCs) hold great promise for the treatment of various human diseases. However, their therapeutic benefits and mechanisms for treating corneal endothelial dysfunction remain undefined. Here, we developed a therapeutic regimen consisting of the combination of hPSC-derived corneal endothelial precursors (CEPs) with nicotinamide (NAM) for effective treatment of corneal endothelial dysfunction. In rabbit and nonhuman primate models, intracameral injection of CEPs and NAM achieved long-term recovery of corneal clarity and thickness, similar with the therapeutic outcome of cultured human corneal endothelial cells (CECs). The transplanted human CEPs exhibited structural and functional integration with host resident CECs. However, the long-term recovery relied on the stimulation of endogenous endothelial regeneration in rabbits, but predominantly on the replacing function of transplanted cells during the 3-year follow-up in nonhuman primates, which resemble human corneal endothelium with limited regenerative capacity. Mechanistically, NAM ensured in vivo proper maturation of transplanted CEPs into functional CECs by preventing premature senescence and endothelial-mesenchymal transition within the TGF-β–enriched aqueous humor. Together, we provide compelling experimental evidence and mechanistic insights of simultaneous delivery of CEPs and NAM as a potential approach for treating corneal endothelial dysfunction.

Anti-VEGF therapy has many limitations that might be resolved by using combination treatment approaches. Here, the authors demonstrate that the dual-targeting of VEGF and PDGF is required for targeting resistant FGF2+ tumors which depend on the recruitment of pericytes on tumor microvessels.

Abstract Background Acute glaucoma, characterized by a sudden elevation in intraocular pressure (IOP) and retinal ganglion cells (RGCs) death, is a major cause of irreversible blindness worldwide that lacks approved effective therapies, validated treatment targets and clear molecular mechanisms. We sought to explore the potential molecular mechanisms underlying the causal link between high IOP and glaucomatous RGCs death. Methods A murine retinal ischemia/ reperfusion (RIR) model and an in vitro oxygen and glucose deprivation/reoxygenation (OGDR) model were used to investigate the pathogenic mechanisms of acute glaucoma. Results Our findings reveal a novel mechanism of microglia-induced pyroptosis-mediated RGCs death associated with glaucomatous vision loss. Genetic deletion of gasdermin D (GSDMD), the effector of pyroptosis, markedly ameliorated the RGCs death and retinal tissue damage in acute glaucoma. Moreover, GSDMD cleavage of microglial cells was dependent on caspase-8 (CASP8)-hypoxia-inducible factor-1α (HIF-1α) signaling. Mechanistically, the newly identified nucleotide-binding leucine-rich repeat-containing receptor (NLR) family pyrin domain-containing 12 (NLRP12) collaborated with NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing protein 4 (NLRC4) downstream of the CASP8-HIF-1α axis, to elicit pyroptotic processes and interleukin-1β (IL-1β) maturation through caspase-1 activation, facilitating pyroptosis and neuroinflammation in acute glaucoma. Interestingly, processing of IL-1β in turn magnified the CASP8-HIF-1α-NLRP12/NLRP3/NLRC4-pyroptosis circuit to accelerate inflammatory cascades. Conclusions These data not only indicate that the collaborative effects of NLRP12, NLRP3 and NLRC4 on pyroptosis are responsible for RGCs death, but also shed novel mechanistic insights into microglial pyroptosis, paving novel therapeutic avenues for the treatment of glaucoma-induced irreversible vision loss through simultaneously targeting of pyroptosis.

Mechanisms regulating the formation of pre-metastatic niches remain poorly understood. Here, the authors show that ITGBL1-containing extracellular vesicles derived from primary colorectal cancer cells activate the production of inflammatory cytokines by resident fibroblasts in distant organs, promoting metastatic cancer growth.

Abstract Recognition of specific antigens expressed in cancer cells is the initial process of cytolytic T cell‐mediated cancer killing. However, this process can be affected by other non‐cancerous cellular components in the tumor microenvironment. Here, it is shown that interleukin‐33 (IL‐33)‐activated macrophages protect melanoma cells from tumor‐infiltrating lymphocyte‐mediated killing. Mechanistically, IL‐33 markedly upregulates metalloprotease 9 (MMP‐9) expression in macrophages, which acts as a sheddase to trim NKG2D, an activating receptor expressed on the surface of natural killer (NK) cells, CD8+ T cells, subsets of CD4+ T cells, iNKT cells, and γδ T cells. Further, MMP‐9 also cleaves the MHC class I molecule, cell surface antigen‐presenting complex molecules, expressed in melanoma cells. Consequently, IL‐33‐induced macrophage MMP‐9 robustly mitigates the tumor killing‐effect by T cells. Genetic and pharmacological loss‐of‐function of MMP‐9 sheddase restore T cell‐mediated cancer killing. Together, these data provide compelling in vitro and in vivo evidence showing novel mechanisms underlying the IL‐33‐macrophage‐MMP‐9 axis‐mediated immune tolerance against cancer cells. Targeting each of these signaling components, including IL‐33 and MMP‐9 provides a new therapeutic paradigm for improving anticancer efficacy by immune therapy.

Abstract The neurokinin‐1 receptor (NK‐1R) antagonists are approved as treatment for chemotherapy‐associated nausea and vomiting in cancer patients. The emerging role of the substance P‐NK‐1R system in oncogenesis raises the possibility of repurposing well‐tolerated NK‐1R antagonists for cancer treatment. This study reports that human colorectal cancer (CRC) patients with high NK‐1R expression have poor survival, and NK‐1R antagonists SR140333 and aprepitant induce apoptotic cell death in CRC cells and inhibit CRC xenograft growth. This cytotoxicity induced by treatment with NK‐1R antagonists is mediated by induction of endoplasmic reticulum (ER) stress. ER stress triggers calcium release, resulting in the suppression of prosurvival extracellular signal‐regulated kinase (ERK)‐c‐Myc signaling. Along with ER calcium release, one ER stress pathway mediated by protein kinase RNA‐like ER kinase (PERK) is specifically activated, leading to increased expression of proapoptotic C/EBP‐homologous protein (CHOP). Moreover, NK‐1R antagonists enhance the efficacy of chemotherapy by increasing the sensitivity and overcoming resistance to 5‐fluorouracil in CRC cells through the induction of sustained ER stress and the consequent suppression of ERK‐c‐Myc signaling both in vitro and in vivo. Collectively, the findings provide novel mechanistic insights into the efficacy of NK‐1R antagonists either as a single agent or in combination with chemotherapy for cancer treatment.

Abstract Effective treatment of patients with severe COVID‐19 to reduce mortality remains one of the most challenging medical issues in controlling unpredictable emergencies caused by the global pandemics. Unfortunately, such effective therapies are not available at this time of writing. In this article, I discuss the possibility of repurposing clinically available anti‐VEGF (vascular endothelial growth factor) drugs that are routinely used in oncology and ophthalmology areas for effective treatment of patients with severe and critical COVID‐19. Our preliminary findings from a clinical trial support the therapeutic concept of using anti‐VEGF for treating patients with severe COVID‐19 to reduce mortality. The aim of this article is to further provide mechanistic insights into the role of VEGF in causing pathological changes during COVID‐19 infection.

Background Indoleamine-2,3-dioxygenase 1 (IDO1) has been intensively pursued as a therapeutic target to reverse the immunosuppressive cancer-immune milieu and promote tumor elimination. However, recent failures of phase III clinical trials with IDO1 inhibitors involved in cancer immunotherapies highlight the urgent need to develop appropriate methods for tracking IDO1 when the cancer-immune milieu is therapeutically modified.Methods We utilized a small-molecule radiotracer, 11C-l-1MTrp, to quantitatively and longitudinally visualize whole-body IDO1 dynamics. Specifically, we first assessed 11C-l-1MTrp in mice-bearing contralateral human tumors with distinct IDO1 expression patterns. Then, we applied 11C-l-1MTrp to longitudinally monitor whole-body IDO1 variations in immunocompetent melanoma-bearing mice treated with 1-methyl-l-tryptophan plus either chemotherapeutic drugs or antibodies targeting programmedcell death 1 and cytotoxic T-lymphocyte-associated protein 4.Results 11C-l-1MTrp positron emission tomography (PET) imaging accurately delineated IDO1 expression in xenograft mouse models. Moreover, we were able to visualize dynamic IDO1 regulation in the mesenteric lymph nodes (MLNs), an off-tumor IDO1 target, where the percentage uptake of 11C-l-1MTrp accurately annotated the therapeutic efficacy of multiple combination immunotherapies in preclinical models. Remarkably, 11C-l-1MTrp signal intensity in the MLNs was inversely related to the specific growth rates of treated tumors, suggesting that IDO1 expression in the MLNs can serve as a new biomarker of the cancer-immune set point.Conclusions PET imaging of IDO1 with 11C-l-1MTrp is a robust method to assess the therapeutic efficacy of multiple combinatorial immunotherapies, improving our understanding of the merit and challenges of IDO1 regimens. Further validation of this animal data in humans is ongoing. We envision that our results will provide a potential precision medicine paradigm for noninvasive visualizing each patient’s individual response in combinatorial cancer immunotherapy, and tailoring optimal personalized combination strategies.

Most COVID-19 victims are old and die from unrelated causes. Here we present twelve complete autopsies, including two rapid autopsies of young patients where the cause of death was COVID-19 ARDS. The main virus induced pathology was in the lung parenchyma and not in the airways. Most coagulation events occurred in the intra-alveolar and not in the intra-vascular space and the few thrombi were mainly composed of aggregated thrombocytes. The dominant inflammatory response was the massive accumulation of CD163 + macrophages and the disappearance of T killer, NK and B-cells. The virus was replicating in the pneumocytes and macrophages but not in bronchial epithelium, endothelium, pericytes or stromal cells. The lung consolidations were produced by a massive regenerative response, stromal and epithelial proliferation and neovascularization. We suggest that thrombocyte aggregation inhibition, angiogenesis inhibition and general proliferation inhibition may have a roll in the treatment of advanced COVID-19 ARDS.

Abstract Antiangiogenic tyrosine kinase inhibitors (AA‐TKIs) have become a promising therapeutic strategy for colorectal cancer (CRC). In clinical practice, a significant proportion of cancer patients temporarily discontinue AA‐TKI treatment due to recurrent toxicities, economic burden or acquired resistance. However, AA‐TKI therapy withdrawal‐induced tumour revascularization frequently occurs, hampering the clinical application of AA‐TKIs. Here, this study demonstrates that tumour perivascular cells mediate tumour revascularization after withdrawal of AA‐TKI therapy. Pharmacological inhibition and genetic ablation of perivascular cells largely attenuate the rebound effect of CRC vascularization in the AA‐TKI cessation experimental settings. Mechanistically, tumour perivascular cell‐derived extracellular vehicles (TPC‐EVs) contain Gas6 that instigates the recruitment of endothelial progenitor cells (EPCs) for tumour revascularization via activating the Axl pathway. Gas6 silence and an Axl inhibitor markedly inhibit tumour revascularization by impairing EPC recruitment. Consequently, combination therapy of regorafenib with the Axl inhibitor improves overall survival in mice metastatic CRC model by inhibiting tumour growth. Together, these data shed new mechanistic insights into perivascular cells in off‐AA‐TKI‐induced tumour revascularization and indicate that blocking the Axl signalling may provide an attractive anticancer approach for sustaining long‐lasting angiostatic effects to improve the therapeutic outcomes of antiangiogenic drugs in CRC.

Summary: The Hippo/Yes-associated protein (YAP) pathway has pivotal roles in innate immune responses against pathogens in macrophages. However, the role of YAP in macrophages during atherosclerosis and its mechanism of YAP activation remain unknown. Here, we find that YAP overexpression in myeloid cells aggravates atherosclerotic lesion size and infiltration of macrophages, whereas YAP deficiency reduces atherosclerotic plaque. Tumor necrosis factor receptor-associated factor 6 (TRAF6), a downstream effector of interleukin-1β (IL-1β), triggers YAP ubiquitination at K252, which interrupts the interaction between YAP and angiomotin and results in enhanced YAP nuclear translocation. The recombinant IL-1 receptor antagonist anakinra reduces atherosclerotic lesion formation, which is abrogated by YAP overexpression. YAP level is increased in human and mouse atherosclerotic vessels, and plasma IL-1β level in patients with STEMI is correlated with YAP protein level in peripheral blood mononuclear cells. These findings elucidate a mechanism of YAP activation, which might be a therapeutic target for atherosclerosis.

Post-cardiac arrest myocardial dysfunction significantly contributes to early mortality after the return of spontaneous circulation. However, no effective therapy is available now. Aldehyde dehydrogenase 2 (ALDH2) enzyme has been shown to protect the heart from aldehyde toxicity such as 4-hydroxy-2-nonenal (4-HNE) and oxidative stress. In this study, we evaluated the effect of enhanced activity or expression of ALDH2 on post-cardiac arrest myocardial dysfunction and survival in a rat cardiac arrest model. Furthermore, we elucidated the underlying mechanisms with a focus on mitochondrial reactive oxygen species (ROS) production in a cell hypoxia/reoxygenation model. A total of 126 rats were used for the ALDH2 activation or cardiac overexpression of ALDH2 studies. Randomization was done 10 min before the respective agonist injection or in vivo gene delivery. We showed that enhanced activity or expression of ALDH2 significantly improved contractile function of the left ventricle and survival rate in rats subjected to cardiac arrest-cardiopulmonary resuscitation procedure. Moreover, ALDH2 prevented cardiac arrest-induced cardiomyocyte death from apoptosis and mitochondrial damage. Mechanistically, 4-HNE, a representative substrate of ALDH2, was dominantly increased in the hypoxia/reoxygenation-exposed cardiomyocytes. Direct addition of 4-HNE led to significantly augmented succinate accumulation and mitochondrial ROS production. Through metabolizing 4-HNE, ALDH2 significantly inhibited mitochondrial ROS production. Our findings provide compelling evidence of the cardioprotective effects of ALDH2 and therapeutic targeting this enzyme would provide an important approach for treating post-cardiac arrest myocardial dysfunction.

Diabetic kidney disease (DKD) is a common cause of end-stage renal disease, and diagnosis and treatment in time can help delay its progress. At present, there are more and more studies on the pathogenesis of DKD; mitochondrial dysfunction plays an important role in DKD. The occurrence and development of DKD is closely related to epigenetic changes and the interaction between mtDNA, ROS, inflammatory factors, and endothelial damage, which continuously aggravates kidney. The change of mtDNA is both the cause of DKD and the result of DKD. It is of great significance to incorporate the change of mtDNA into the monitoring of patients with diabetes. Existing evidence indicates that changes in mtDNA copy number in blood and urine reflect mitochondrial dysfunction and the severity of DKD. However, large-scale, long-term follow-up clinical trials are still needed to determine the threshold range. By the time, mitochondrial-targeted antioxidants will become a new method for the treatment of DKD and other diabetic complications; mtDNA also can be a therapeutic target for them.

White adipocytes can be stimulated to express thermogenic genes in a process known as beiging. Here, the authors show that miR-327 is downregulated during beiging, which releases FGF10 from inhibition and supports beige adipocyte formation via signaling through FGFR2.

Abstract Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti‐angiogenic treatment has limited efficacy due to therapy‐induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy‐induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid‐derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti‐angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor‐induced metastases, and high Apelin levels correlate with poor prognosis of anti‐angiogenic therapy patients. These data identify a druggable anti‐angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases.

Anti-VEGF therapy often produces limited beneficial effects in cancer patients. Here, the authors show that discontinuation of anti-VEGF cancer therapy in xenografts-bearing mice increases cancer cells extravasation and intravasation in liver through the host-derived VEGF.

Cold-induced activation of thermogenesis in white adipose tissue (WAT), or ‘beiging’, is associated with WAT angiogenesis. Here the authors show that PDGF-CC is secreted from endothelial cells in the context of WAT angiogenesis and its paracrine action on adipocytes contributes to cold-induced beiging.

Elevated IL-33 levels have been correlated with metastasis and poor prognosis. Here the authors show in mouse tumour xenograft models that PDGF-BB produced by tumour cells induces IL-33 via Sox7 in tumour pericytes, and IL-33 promotes metastasis through its effects on tumour-associated macrophages.

Blocking lymphangiogenesis holds great therapeutic potential, however, only few inhibitors are known. Here the authors show that membrane type 1-matrix metalloproteinase suppresses lymphangiogenesis by curbing both LYVE-1-mediated proliferation of lymphatic endothelial cells and production of lymphangiogenic factors by macrophages.

Abstract In human patients, drugs that block tumor vessel growth are widely used to treat a variety of cancer types. Many rigorous phase 3 clinical trials have demonstrated significant survival benefits; however, the addition of an anti-angiogenic component to conventional therapeutic modalities has generally produced modest survival benefits for cancer patients. Currently, it is unclear why these clinically available drugs targeting the same angiogenic pathways produce dissimilar effects in preclinical models and human patients. In this article, we discuss possible mechanisms of various anti-angiogenic drugs and the future development of optimized treatment regimens.

Only half of patients with relapsed B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) currently survive with standard treatment protocols. Predicting individual patient responses to defined drugs prior to application would help therapy stratification and could improve survival. With the purpose to aid personalized targeted treatment approaches, we developed a human–zebrafish xenograft (ALL-ZeFiX) assay to predict drug response in a patient in 5 days. Leukemia blast cells were pericardially engrafted into transiently immunosuppressed Danio rerio embryos, and engrafted embryos treated for the test case, venetoclax, before single-cell dissolution for quantitative whole blast cell analysis. Bone marrow blasts from patients with newly diagnosed or relapsed BCP-ALL were successfully expanded in 60% of transplants in immunosuppressed zebrafish embryos. The response of BCP-ALL cell lines to venetoclax in ALL-ZeFiX assays mirrored responses in 2D cultures. Venetoclax produced varied responses in patient-derived BCP-ALL grafts, including two results mirroring treatment responses in two refractory BCP-ALL patients treated with venetoclax. Here we demonstrate proof-of-concept for our 5-day ALL-ZeFiX assay with primary patient blasts and the test case, venetoclax, which after expanded testing for further targeted drugs could support personalized treatment decisions within the clinical time window for decision-making.

Molecular mechanisms underlying tumor VEGF-induced host anemia and bone marrow cell (BMC) mobilization remain unknown. Here, we report that tumor VEGF markedly induced sinusoidal vasculature dilation in bone marrow (BM) and BMC mobilization to tumors and peripheral tissues in mouse and human tumor models. Unexpectedly, anti-VEGFR2, but not anti-VEGFR1, treatment completely blocked VEGF-induced anemia and BMC mobilization. Genetic deletion of Vegfr2 in endothelial cells markedly ablated VEGF-stimulated BMC mobilization. Conversely, deletion of the tyrosine kinase domain from Vegfr1 gene (Vegfr1TK−/−) did not affect VEGF-induced BMC mobilization. Analysis of VEGFR1+/VEGFR2+ populations in peripheral blood and BM showed no significant ratio difference between VEGF- and control tumor-bearing animals. These findings demonstrate that vascular dilation through the VEGFR2 signaling is the mechanism underlying VEGF-induced BM mobilization and anemia. Thus, our data provide mechanistic insights on VEGF-induced BMC mobilization in tumors and have therapeutic implications by targeting VEGFR2 for cancer therapy.

The p53 upregulated modulator of apoptosis (PUMA) is known as an essential apoptosis inducer. Here, we report the seemingly paradoxical finding that PUMA is a proangiogenic factor critically required for the proliferation and survival of vascular and microglia cells. Strikingly, Puma deficiency by genetic deletion or small hairpin RNA knockdown inhibited developmental and pathological angiogenesis and reduced microglia numbers in vivo, whereas Puma gene delivery increased angiogenesis and cell survival. Mechanistically, we revealed that PUMA plays a critical role in regulating autophagy by modulating Erk activation and intracellular calcium level. Our findings revealed an unexpected function of PUMA in promoting angiogenesis and warrant more careful investigations into the therapeutic potential of PUMA in treating cancer and degenerative diseases.

Molecular mechanisms underlying circadian-regulated physiological processes remain largely unknown. Here, we show that disruption of the circadian clock by both constant exposure to light and genetic manipulation of key genes in zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. Using a promoter-reporter system consisting of various deleted vegf-promoter mutants, we show that Bmal1 directly binds to and activates the vegf promoter via E-boxes. Additionally, we provide evidence that knockdown of Bmal1 leads to impaired Notch-inhibition-induced vascular sprouting. These results shed mechanistic insight on the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis.

Organisms need to adapt to the ecological constraints in their habitat. How specific processes reflect such adaptations are difficult to model experimentally. We tested whether environmental shifts in oxygen tension lead to events in the adult newt brain that share features with processes occurring during neuronal regeneration under normoxia. By experimental simulation of varying oxygen concentrations, we show that hypoxia followed by re-oxygenation lead to neuronal death and hallmarks of an injury response, including activation of neural stem cells ultimately leading to neurogenesis. Neural stem cells accumulate reactive oxygen species (ROS) during re-oxygenation and inhibition of ROS biosynthesis counteracts their proliferation as well as neurogenesis. Importantly, regeneration of dopamine neurons under normoxia also depends on ROS-production. These data demonstrate a role for ROS-production in neurogenesis in newts and suggest that this role may have been recruited to the capacity to replace lost neurons in the brain of an adult vertebrate.

Background and objective VEGF plays an important role in the development of cancer. The aim of this study is to observe the structural alterations in multiple organs of high VEGF expression mice, and insight into the role of tumor-derived VEGF in the development of CASS. Methods Murine fibrosarcoma of T241-VEGF and T241- Vector tumor cells were transplanted subcutaneously in mice to construct the xenograft tumor model. The mice gross examinations were observed and the percentage of survival animals in each group is presented. The level of hemoglobin, the numbers of erythrocytes and serum concentration of VEGF in peripheral blood were analyzed. Histological analysis of liver, spleen, adrenal gland and bone-marrow were applied. Vascular networks in tumors were analyzed under a confocal microscope. Results The VEGF-expressing tumor bearing mice manifested CASS by severe anemia, hepatosplenomegaly and loss of body weight. The survival rate of mice was decreased. The level of hemoglobin and erythrocytes in circulating blood were significantly reduced (P

Ocular neovascularisation is a pathological hallmark of some forms of debilitating blindness including diabetic retinopathy, age related macular degeneration and retinopathy of prematurity. Current therapies for delaying unwanted ocular angiogenesis include laser surgery or molecular inhibition of the pro-angiogenic factor VEGF. However, targeting of angiogenic pathways other than, or in combination to VEGF, may lead to more effective and safer inhibitors of intraocular angiogenesis. In a small chemical screen using zebrafish, we identify LY294002 as an effective and selective inhibitor of both developmental and ectopic hyaloid angiogenesis in the eye. LY294002, a PI3 kinase inhibitor, exerts its anti-angiogenic effect in a dose-dependent manner, without perturbing existing vessels. Significantly, LY294002 delivered by intraocular injection, significantly inhibits ocular angiogenesis without systemic side-effects and without diminishing visual function. Thus, targeting of PI3 kinase pathways has the potential to effectively and safely treat neovascularisation in eye disease.

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs.

Optic neuropathies, characterized by injury of retinal ganglion cell (RGC) axons of the optic nerve, cause incurable blindness worldwide. Mesenchymal stem cell–derived small extracellular vesicles (MSC-sEVs) represent a promising “cell-free” therapy for regenerative medicine; however, the therapeutic effect on neural restoration fluctuates, and the underlying mechanism is poorly understood. Here, we illustrated that intraocular administration of MSC-sEVs promoted both RGC survival and axon regeneration in an optic nerve crush mouse model. Mechanistically, MSC-sEVs primarily targeted retinal mural cells to release high levels of colony-stimulating factor 3 (G-CSF) that recruited a neural restorative population of Ly6Clow monocytes/monocyte-derived macrophages (Mo/MΦ). Intravitreal administration of G-CSF, a clinically proven agent for treating neutropenia, or donor Ly6Clow Mo/MΦ markedly improved neurological outcomes in vivo. Together, our data define a unique mechanism of MSC-sEV-induced G-CSF-to-Ly6Clow Mo/MΦ signaling in repairing optic nerve injury and highlight local delivery of MSC-sEVs, G-CSF, and Ly6Clow Mo/MΦ as therapeutic paradigms for the treatment of optic neuropathies [ABSTRACT FROM AUTHOR]