A therapy for suppressing canonical and noncanonical SARS-CoV-2 viral entry and an intrinsic intrapulmonary inflammatory response.

The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.

Novel formylpeptide receptor 1/2 agonist limits hypertension-induced cardiovascular damage.

AIMS: Formylpeptide receptors (FPRs) play a critical role in the regulation of inflammation, an important driver of hypertension-induced end-organ damage. We have previously reported that the biased FPR small-molecule agonist, compound17b (Cmpd17b), is cardioprotective against acute, severe inflammatory insults. Here, we reveal the first compelling evidence of the therapeutic potential of this novel FPR agonist against a longer-term, sustained inflammatory insult, i.e. hypertension-induced end-organ damage. The parallels between the murine and human hypertensive proteome were also investigated. METHODS AND RESULTS: The hypertensive response to angiotensin II (Ang II, 0.7 mg/kg/day, s.c.) was attenuated by Cmpd17b (50 mg/kg/day, i.p.). Impairments in cardiac and vascular function assessed via echocardiography were improved by Cmpd17b in hypertensive mice. This functional improvement was accompanied by reduced cardiac and aortic fibrosis and vascular calcification. Cmpd17b also attenuated Ang II-induced increased cardiac mitochondrial complex 2 respiration. Proteomic profiling of cardiac and aortic tissues and cells, using label-free nano-liquid chromatography with high-sensitivity mass spectrometry, detected and quantified ∼6000 proteins. We report hypertension-impacted protein clusters associated with dysregulation of inflammatory, mitochondrial, and calcium responses, as well as modified networks associated with cardiovascular remodelling, contractility, and structural/cytoskeletal organization. Cmpd17b attenuated hypertension-induced dysregulation of multiple proteins in mice, and of these, ∼110 proteins were identified as similarly dysregulated in humans suffering from adverse aortic remodelling and cardiac hypertrophy. CONCLUSION: We have demonstrated, for the first time, that the FPR agonist Cmpd17b powerfully limits hypertension-induced end-organ damage, consistent with proteome networks, supporting development of pro-resolution FPR-based therapeutics for treatment of systemic hypertension complications.

The oxytocin antagonist cligosiban reduces human prostate contractility: Implications for the treatment of benign prostatic hyperplasia.

BACKGROUND AND PURPOSE: With increasing life expectancy, benign prostatic hyperplasia (BPH) consequently affects more ageing men, illustrating the urgent need for advancements in BPH therapy. One emerging possibility may be the use of oxytocin antagonists to relax smooth muscle cells in the prostate, similar to the currently used (although often associated with side effects) α1-adrenoceptor blockers. EXPERIMENTAL APPROACH: For the first time we used live-imaging, combined with a novel image analysis method, to investigate the multidirectional contractions of the human prostate and determine their changes in response to oxytocin and the oxytocin antagonists atosiban and cligosiban. Human prostate samples were obtained and compared from patients undergoing prostatectomy due to prostate cancer as well as from patients with transurethral resection of prostate tissue due to severe BPH. KEY RESULTS: The two cohorts of tissue samples showed spontaneous multidirectional contractions, which significantly increased after the addition of oxytocin. Different to atosiban, which showed ambiguous effects of short duration, only long-acting cligosiban reliably prevented, as well as counteracted, any contractile oxytocin effect. Furthermore, cligosiban visibly reduced not only oxytocin-induced contractions, but also showed intrinsic activity to relax prostatic tissue. CONCLUSION AND IMPLICATIONS: Thus, the oxytocin antagonist cligosiban could be an interesting candidate in the search for novel BPH treatment options.

Impact of conjugation to different lipids on the lymphatic uptake and biodistribution of brush PEG polymers.

Delivery to peripheral lymphatics can be achieved following interstitial administration of nano-sized delivery systems (nanoparticles, liposomes, dendrimers etc) or molecules that hitchhike on endogenous nano-sized carriers (such as albumin). The published work concerning the hitchhiking approach has mostly focussed on the lymphatic uptake of vaccines conjugated directly to albumin binding moieties (ABMs such as lipids, Evans blue dye derivatives or peptides) and their subsequent trafficking into draining lymph nodes. The mechanisms underpinning access and transport of these constructs into lymph fluid, including potential interaction with other endogenous nanocarriers such as lipoproteins, have largely been ignored. Recently, we described a series of brush polyethylene glycol (PEG) polymers containing end terminal short-chain or medium-chain hydrocarbon tails (1C2 or 1C12, respectively), cholesterol moiety (Cho), or medium-chain or long-chain diacylglycerols (2C12 or 2C18, respectively). We evaluated the association of these materials with albumin and lipoprotein in rat plasma, and their intravenous (IV) and subcutaneous (SC) pharmacokinetic profiles. Here we fully detail the association of this suite of polymers with albumin and lipoproteins in rat lymph, which is expected to facilitate lymph transport of the materials from the SC injection site. Additionally, we characterise the thoracic lymph uptake, tissue and lymph node biodistribution of the lipidated brush PEG polymers following SC administration to thoracic lymph cannulated rats. All polymers had moderate lymphatic uptake in rats following SC dosing with the lymph uptake higher for 1C2-PEG, 2C12-PEG and 2C18-PEG (5.8%, 5.9% and 6.7% dose in lymph, respectively) compared with 1C12-PEG and Cho-PEG (both 1.5% dose in lymph). The enhanced lymph uptake of 1C2-PEG, 2C12-PEG and 2C18-PEG appeared related to their association profile with different lipoproteins. The five polymers displayed different biodistribution patterns in major organs and tissues in mice. All polymers reached immune cells deep within the inguinal lymph nodes of mice following SC dosing. The ability to access these immune cells suggests the potential of the polymers as platforms for the delivery of vaccines and immunotherapies. Future studies will focus on evaluating the lymphatic targeting and therapeutic potential of drug or vaccine-loaded polymers in pre-clinical disease models.

Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

The pro-resolving mediator, annexin A1 regulates blood pressure, and age-associated changes in cardiovascular function and remodeling.

Aging is associated with chronic, low-level inflammation which may contribute to cardiovascular pathologies such as hypertension and atherosclerosis. This chronic inflammation may be opposed by endogenous mechanisms to limit inflammation, for example, by the actions of annexin A1 (ANXA1), an endogenous glucocorticoid-regulated protein that has anti-inflammatory and pro-resolving activity. We hypothesized the pro-resolving mediator ANXA1 protects against age-induced changes in blood pressure (BP), cardiovascular structure and function, and cardiac senescence. BP was measured monthly in conscious mature (4-month) and middle-aged (12-month) ANXA1-deficient (ANXA1-/- ) and wild-type C57BL/6 mice. Body composition was measured using EchoMRI, and both cardiac and vascular function using ultrasound imaging. Cardiac hypertrophy, fibrosis and senescence, vascular fibrosis, elastin, and calcification were assessed histologically. Gene expression relevant to structural remodeling, inflammation, and cardiomyocyte senescence were also quantified. In C57BL/6 mice, progression from 4 to 12 months of age did not affect the majority of cardiovascular parameters measured, with the exception of mild cardiac hypertrophy, vascular calcium, and collagen deposition. Interestingly, ANXA1-/- mice exhibited higher BP, regardless of age. Additionally, age progression had a marked impact in ANXA1-/- mice, with markedly augmented vascular remodeling, impaired vascular distensibility, and body composition. Consistent with vascular dysfunction, cardiac dysfunction, and hypertrophy were also evident, together with markers of senescence and inflammation. These findings suggest that endogenous ANXA1 plays a critical role in regulating BP, cardiovascular function, and remodeling and delays cardiac senescence. Our findings support the development of novel ANXA1-based therapies to prevent age-related cardiovascular pathologies.

Sex-Dependent Attentional Impairments in a Subchronic Ketamine Mouse Model for Schizophrenia.

Background: The development of more effective treatments for schizophrenia targeting cognitive and negative symptoms has been limited, partly due to a disconnect between rodent models and human illness. Ketamine administration is widely used to model symptoms of schizophrenia in both humans and rodents. In mice, subchronic ketamine treatment reproduces key dopamine and glutamate dysfunction; however, it is unclear how this translates into behavioral changes reflecting positive, negative, and cognitive symptoms. Methods: In male and female mice treated with either subchronic ketamine or saline, we assessed spontaneous and amphetamine-induced locomotor activity to measure behaviors relevant to positive symptoms, and used a touchscreen-based progressive ratio task of motivation and the rodent continuous performance test of attention to capture specific negative and cognitive symptoms, respectively. To explore neuronal changes underlying the behavioral effects of subchronic ketamine treatment, we quantified expression of the immediate early gene product, c-Fos, in key corticostriatal regions using immunofluorescence. Results: We showed that spontaneous locomotor activity was unchanged in male and female subchronic ketamine-treated animals, and amphetamine-induced locomotor response was reduced. Subchronic ketamine treatment did not alter motivation in either male or female mice. In contrast, we identified a sex-specific effect of subchronic ketamine on attentional processing wherein female mice performed worse than control mice due to increased nonselective responding. Finally, we showed that subchronic ketamine treatment increased c-Fos expression in prefrontal cortical and striatal regions, consistent with a mechanism of widespread disinhibition of neuronal activity. Conclusions: Our results highlight that the subchronic ketamine mouse model reproduces a subset of behavioral symptoms that are relevant for schizophrenia.

Distribution of smooth muscle actin and collagen in superficial peritoneal endometriotic lesions varies from the surrounding microenvironment.

RESEARCH QUESTION: Do different subtypes of superficial peritoneal endometriotic lesions exist, based on the presence and morphology of smooth muscle, collagen fibres and immune cell populations? DESIGN: A retrospective cohort study of 24 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Immunofluorescence was used to delineate the CD10 stromal area of lesions (n = 271 lesions from 67 endometriotic biopsies), and then smooth muscle actin (SMA) positive tissue and immune cell populations (CD45+ and CD68+) were quantified within and adjacent to these lesions. Second harmonic generation microscopy was used to evaluate the presence and morphology of type-1 collagen fibres within and surrounding lesions. RESULTS: Overall, immune cell numbers and the area of SMA and collagen within endometriotic lesions tended to be low, but a spectrum of presentations significantly varied, particularly in the adjacent tissue microenvironment, based on lesion locations, the morphology of endometriotic gland profiles, or both. Lesions in which collagen fibres formed well aligned capsules around the CD10+ stromal border were identified compared with lesions in which collagen fibre distribution was random. Considerable inter- and intra-patient variability in the morphology of SMA and collagen was observed within and surrounding lesions. CONCLUSION: These data demonstrate considerable diversity in the presence of immune cells and morphology of SMA and collagen within, but even more so, surrounding endometriotic lesions, even within individual patients. This heterogeneity, especially within individual patients, presents a challenge to incorporating these cell and tissue types into any new endometriosis classification systems or prognostic approaches.

The Arteria Lymphatica and Lymphatic Microperforators: A Dedicated Blood Supply to Collecting Lymphatics and Their Potential Implications in Lymphedema: Anatomical Description.

Background: Lymphedema is common after lymphatic damage in cancer treatment, with negative impacts on function and quality of life. Evidence suggests that blood vessel microvasculature is sensitive to irradiation and trauma; however, despite knowledge regarding dedicated mural blood supply to arteries and veins (vasa vasorum), equivalent blood vessels supplying lymphatics have not been characterized. We studied collecting lymphatics for dedicated mural blood vessels in our series of 500 lymphaticovenous anastomosis procedures for lymphedema, and equivalent controls. Methods: Microscopic images of lymphatics from lymphedema and control patients were analyzed for lymphatic wall vascular density. Collecting lymphatics from 20 patients with lymphedema and 10 control patients were sampled for more detailed analysis (podoplanin immunostaining, light/confocal microscopy, microcomputed tomography, and transmission electron microscopy) to assess lymphatic wall ultrastructure and blood supply. Results: Analysis revealed elaborate, dense blood microvessel networks associating with lymphatic walls in lymphedema patients and smaller equivalent vessels in controls. These vasa vasora or "arteria lymphatica" were supplied by regular axial blood vessels, parallel to lymphatic microperforators linking dermal and collecting lymphatics. Lymphatic walls were thicker in lymphedema patients than controls, with immunohistochemistry, computed tomography, transmission electron microscopy, and confocal microscopy characterizing abnormal blood vessels (altered appearance, thickened walls, elastin loss, narrow lumina, and fewer red blood cells) on these lymphatic walls. Conclusions: Dedicated blood vessels on lymphatics are significantly altered in lymphedema. A better understanding of the role of these vessels may reveal mechanistic clues into lymphedema pathophysiology and technical aspects of lymphedema microsurgery, and suggest potential novel therapeutic targets.

TRPV4 is expressed by enteric glia and muscularis macrophages of the colon but does not play a prominent role in colonic motility.

Background: Mechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution. Methods: In this study we profiled functional expression of TRPV4 using Ca2+ imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessed in vitro using videomapping and contraction assays. Results: The TRPV4 agonist GSK1016790A evoked Ca2+ signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFRα+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+ responses by a subpopulation of glial cells, followed by sustained Ca2+ signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+ release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examined in vitro. Conclusions: We reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility.

Functionalisation of brush polyethylene glycol polymers with specific lipids extends their elimination half-life through association with natural lipid trafficking pathways.

Polymeric prodrugs have been applied to control the delivery of various types of therapeutics. Similarly, conjugation of peptide therapeutics to lipids has been used to prolong systemic exposure. Here, we extend on these two approaches by conjugating brush polyethylene glycol (PEG) polymers with different lipid components including short-chain (1C2) or medium-chain (1C12) monoalkyl hydrocarbon tails, cholesterol (Cho), and diacylglycerols composed of two medium-chain (2C12) or long-chain (2C18) fatty acids. We uniquely evaluate the integration of these lipid-polymers into endogenous lipid trafficking pathways (albumin and lipoproteins) and the impact of lipid conjugation on plasma pharmacokinetics after intravenous (IV) and subcutaneous (SC) dosing to cannulated rats. The IV and SC elimination half-lives of Cho-PEG (13 and 22 h, respectively), 2C12-PEG (11 and 17 h, respectively) and 2C18-PEG (12 h for both) were prolonged compared to 1C2-PEG (3 h for both) and 1C12-PEG (4 h for both). Interestingly, 1C2-PEG and 1C12-PEG had higher SC bioavailability (40 % and 52 %, respectively) compared to Cho-PEG, 2C12-PEG and 2C18-PEG (25 %, 24 % and 23 %, respectively). These differences in pharmacokinetics may be explained by the different association patterns of the polymers with rat serum albumin (RSA), bovine serum albumin (BSA) and lipoproteins. For example, in pooled plasma (from IV pharmacokinetic studies), 2C18-PEG had the highest recovery in the high-density lipoprotein (HDL) fraction. In conclusion, the pharmacokinetics of brush PEG polymers can be tuned via conjugation with different lipids, which can be utilised to tune the elimination half-life, biodistribution and effect of therapeutics for a range of medical applications. STATEMENT OF SIGNIFICANCE: Lipidation of therapeutics such as peptides has been employed to extend their plasma half-life by promoting binding to serum albumin, providing protection against rapid clearance. Here we design and evaluate innovative biomaterials consisting of brush polyethylene glycol polymers conjugated with different lipids. Importantly, we show for the first time that lipidated polymeric materials associate with endogenous lipoprotein trafficking pathways and this, in addition to albumin binding, controls their plasma pharmacokinetics. We find that conjugation to dialkyl lipids and cholesterol leads to higher association with lipid trafficking pathways, and more sustained plasma exposure, compared to conjugation to short and monoalkyl lipids. Our lipidated polymers can thus be utilised as delivery platforms to tune the plasma half-life of various pharmaceuticals.

Orexin 2 receptor antagonism sex-dependently improves sleep/wakefulness and cognitive performance in tau transgenic mice.

BACKGROUND AND PURPOSE: Tau pathology contributes to a bidirectional relationship between sleep disruption and neurodegenerative disease. Tau transgenic rTg4510 mice model tauopathy symptoms, including sleep/wake disturbances, which manifest as marked hyperarousal. This phenotype can be prevented by early transgene suppression; however, whether hyperarousal can be rescued after onset is unknown. EXPERIMENTAL APPROACH: Three 8-week experiments were conducted with wild-type and rTg4510 mice after age of onset of hyperarousal (4.5 months): (1) Tau transgene suppression with doxycycline (200 ppm); (2) inactive phase rapid eye movement (REM) sleep enhancement with the dual orexin receptor antagonist suvorexant (50 mg·kg-1 ·day-1 ); or (3) Active phase non-NREM (NREM) and REM sleep enhancement using the selective orexin 2 (OX2 ) receptor antagonist MK-1064 (40 mg·kg-1 ·day-1 ). Sleep was assessed using polysomnography, cognition using the Barnes maze, and tau pathology using immunoblotting and/or immunohistochemistry. KEY RESULTS: Tau transgene suppression improved tauopathy and hippocampal-dependent spatial memory, but did not modify hyperarousal. Pharmacological rescue of REM sleep deficits did not improve spatial memory or tau pathology. In contrast, normalising hyperarousal by increasing both NREM and REM sleep via OX2 receptor antagonism restored spatial memory, independently of tauopathy, but only in male rTg4510 mice. OX2 receptor antagonism induced only short-lived hypnotic responses in female rTg4510 mice and did not improve spatial memory, indicating a tau- and sex-dependent disruption of OX2 receptor signalling. CONCLUSIONS AND IMPLICATIONS: Pharmacologically reducing hyperarousal corrects tau-induced sleep/wake and cognitive deficits. Tauopathy causes sex-dependent disruptions of OX2 receptor signalling/function, which may have implications for choice of hypnotic therapeutics in tauopathies.

NET formation is a default epigenetic program controlled by PAD4 in apoptotic neutrophils.

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosis-induced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils.

Therapeutic blockade of ER stress and inflammation prevents NASH and progression to HCC.

The incidence of hepatocellular carcinoma (HCC) is rapidly rising largely because of increased obesity leading to nonalcoholic steatohepatitis (NASH), a known HCC risk factor. There are no approved treatments to treat NASH. Here, we first used single-nucleus RNA sequencing to characterize a mouse model that mimics human NASH-driven HCC, the MUP-uPA mouse fed a high-fat diet. Activation of endoplasmic reticulum (ER) stress and inflammation was observed in a subset of hepatocytes that was enriched in mice that progress to HCC. We next treated MUP-uPA mice with the ER stress inhibitor BGP-15 and soluble gp130Fc, a drug that blocks inflammation by preventing interleukin-6 trans-signaling. Both drugs have progressed to phase 2/3 human clinical trials for other indications. We show that this combined therapy reversed NASH and reduced NASH-driven HCC. Our data suggest that these drugs could provide a potential therapy for NASH progression to HCC.

Small extracellular vesicles promote invadopodia activity in glioblastoma cells in a therapy-dependent manner.

PURPOSE: The therapeutic efficacy of radiotherapy/temozolomide treatment for glioblastoma (GBM) is limited by the augmented invasiveness mediated by invadopodia activity of surviving GBM cells. As yet, however the underlying mechanisms remain poorly understood. Due to their ability to transport oncogenic material between cells, small extracellular vesicles (sEVs) have emerged as key mediators of tumour progression. We hypothesize that the sustained growth and invasion of cancer cells depends on bidirectional sEV-mediated cell-cell communication. METHODS: Invadopodia assays and zymography gels were used to examine the invadopodia activity capacity of GBM cells. Differential ultracentrifugation was utilized to isolate sEVs from conditioned medium and proteomic analyses were conducted on both GBM cell lines and their sEVs to determine the cargo present within the sEVs. In addition, the impact of radiotherapy and temozolomide treatment of GBM cells was studied. RESULTS: We found that GBM cells form active invadopodia and secrete sEVs containing the matrix metalloproteinase MMP-2. Subsequent proteomic studies revealed the presence of an invadopodia-related protein sEV cargo and that sEVs from highly invadopodia active GBM cells (LN229) increase invadopodia activity in sEV recipient GBM cells. We also found that GBM cells displayed increases in invadopodia activity and sEV secretion post radiation/temozolomide treatment. Together, these data reveal a relationship between invadopodia and sEV composition/secretion/uptake in promoting the invasiveness of GBM cells. CONCLUSIONS: Our data indicate that sEVs secreted by GBM cells can facilitate tumour invasion by promoting invadopodia activity in recipient cells, which may be enhanced by treatment with radio-chemotherapy. The transfer of pro-invasive cargos may yield important insights into the functional capacity of sEVs in invadopodia.

Beta-blockade enhances anthracycline control of metastasis in triple-negative breast cancer.

Beta-adrenergic blockade has been associated with improved cancer survival in patients with triple-negative breast cancer (TNBC), but the mechanisms of these effects remain unclear. In clinical epidemiological analyses, we identified a relationship between beta-blocker use and anthracycline chemotherapy in protecting against TNBC progression, disease recurrence, and mortality. We recapitulated the effect of beta-blockade on anthracycline efficacy in xenograft mouse models of TNBC. In metastatic 4T1.2 and MDA-MB-231 mouse models of TNBC, beta-blockade improved the efficacy of the anthracycline doxorubicin by reducing metastatic development. We found that anthracycline chemotherapy alone, in the absence of beta-blockade, increased sympathetic nerve fiber activity and norepinephrine concentration in mammary tumors through the induction of nerve growth factor (NGF) by tumor cells. Moreover, using preclinical models and clinical samples, we found that anthracycline chemotherapy up-regulated ß2-adrenoceptor expression and amplified receptor signaling in tumor cells. Neurotoxin inhibition of sympathetic neural signaling in mammary tumors using 6-hydroxydopamine or genetic deletion of NGF or ß2-adrenoceptor in tumor cells enhanced the therapeutic effect of anthracycline chemotherapy by reducing metastasis in xenograft mouse models. These findings reveal a neuromodulatory effect of anthracycline chemotherapy that undermines its potential therapeutic impact, which can be overcome by inhibiting ß2-adrenergic signaling in the tumor microenvironment. Supplementing anthracycline chemotherapy with adjunctive ß2-adrenergic antagonists represents a potential therapeutic strategy for enhancing the clinical management of TNBC.

A novel device for investigating structure-function relationships and mechanoadaptation of biological tissues.

Exploring the structure-function relationships of cartilage on a microstructural level is crucial for tissue engineering approaches aiming to restore function. Therefore, a combination of mechanical testing with cell and tissue-level imaging would allow for longitudinal studying loading mechanisms, biological responses and mechanoadaptation of tissues at a microstructural level. This paper describes the design and validation of FELIX, a custom-built device for non-destructive image-guided micromechanical evaluation of biological tissues and tissue-engineered constructs. It combines multiphoton microscopy with non-destructive mechanical testing of native soft tissues. Ten silicone samples of the same size were mechanically tested with FELIX by different users to assess the repeatability and reproducibility. The results indicate that FELIX can successfully substitute mechanical testing protocols with a commercial device without compromising precision. Furthermore, FELIX demonstrated consistent results across repeated measurements, with very small deviations. Therefore, FELIX can be used to accurately measure biomechanical properties by different users for separate studies. Additionally, cell nuclei and collagen of porcine articular cartilage were successfully imaged under compression. Cell viability remained high in chondrocytes cultured in agarose over 21 days. Furthermore, there were no signs of contamination indicating a cell friendly, sterile environment for longitudinal studies. In conclusion, this work demonstrates that FELIX can consistently quantify mechanical measures without compromising precision. Furthermore, it is biocompatible allowing for longitudinal measurements.

Intra-articular Injection of a B Cell Depletion Antibody Enhances Local Exposure to the Joint-Draining Lymph Node in Mice with Collagen-Induced Arthritis.

Changes to the number, type, and function of immune cells within the joint-draining lymphatics is a major contributor to the progression of inflammatory arthritis. In particular, there is a significant expansion in pathogenic B cells in the joint-draining lymph node (jdLN). These B cells appear to clog the lymphatic sinuses in the lymph node, inhibit lymph flow, and therefore, reduce the clearance of inflammatory fluid and cells from the joint. Taken together, there is potential to treat inflammatory arthritis more effectively, as well as reduce off-target side effects, with localized delivery of B-cell depleting therapies to the jdLNs. We recently reported that joint-draining lymphatic exposure of biologic disease-modifying anti-rheumatic drugs (DMARDs), including the B cell depletion antibody rituximab, is increased in healthy rats following intra-articular (IA) compared to subcutaneous (SC) or intravenous (IV) administration. This suggests that IA administration of B cell depleting antibodies may increase delivery to target cells in the jdLN and increase the effectiveness of B cell depletion compared to standard SC or IV administration. However, whether enhanced local delivery of DMARDs to the jdLN is also achieved after IA injection in the setting of inflammatory arthritis, where there is inflammation in the joint and jdLN B cell expansion is unknown. We, therefore, assessed the lymph node distribution, absorption and plasma pharmacokinetics, and B cell depletion at different sites after IA, SC, or IV administration of a fluorescently labeled mouse anti-CD20 B cell depleting antibody (Cy5-αCD20) in healthy mice compared to mice with collagen-induced arthritis (CIA). The absorption and plasma pharmacokinetics of Cy5-αCD20 appeared unaltered in mice with CIA whereas distribution of Cy5-αCD20 to the jdLNs was generally increased in mice with CIA, regardless of the route of administration. However, IA administration led to greater and more specific exposure to the jdLNs. Consistent with increased Cy5-αCD20 in the jdLNs of CIA compared to healthy mice, there was a greater reduction in jdLN weight and a trend toward greater jdLN B cell depletion at 24 h compared to 4 h after IA compared to SC and IV administration. Taken together, this data supports the potential to improve local efficacy of B cell depletion therapies through a jdLN-directed approach which will enable a reduction in dose and systemic toxicities.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

Systematic Investigation of Metabolic Oligosaccharide Engineering Efficiency in Intestinal Cells Using a Dibenzocyclooctyne-Monosaccharide Conjugate.

Metabolic oligosaccharide engineering (MOE) of cells with synthetic monosaccharides can introduce functionality to the glycans of cell membranes. Unnatural sugars (e. g., peracetylated mannose-azide) can be expressed on the cell surface with the azide group in place. After MOE, the azide group can participate in a copper-free click reaction with an alkyne (e. g., dibenzocyclooctyne, DBCO) probe. This allows the metabolic fate of monosaccharides in cells to be understood. However, in a drug delivery context it is desirable to have azide groups on the probe (e. g. a drug delivery particle) and the alkyne (e. g. DBCO) on the cell surface. Consequently, the labelling efficiency of intestinal cell lines (Caco-2 and HT29-MTX-E12) treated with N-dibenzocyclooctyne-tetra-acetylmannosamine, and the concentration- and time-dependent labelling were determined. Furthermore, the labelling of mucus in HT29-MTX-E12 cells with DBCO was shown. This study highlights the potential for using MOE to target azide-functionalised probes to intestinal tissues for drug delivery applications.