Increased Expression Of ICAM-1 Research Articles

Role of ICAM-1 in the Adhesion of T Cells to Enteric Glia: Perspectives in the Formation of Plexitis in Crohn’s Disease

Background & AimsThe presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn’s disease. To decipher the mechanisms leading to their formation, T cell interactions with enteric neural cells were studied in vitro and in vivo. MethodsT cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn’s disease. The mechanisms involved in T cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro, but also in vivo in Winnie mice. ResultsThe mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn’s disease (2.42+/-0.5) as compared to controls (0.36+/-0.08, p=0.0007). Co-culture experiments showed that exposure to pro-inflammatory cytokines enhanced T cell adhesion to glia and increased ICAM-1 expression in glia. We next demonstrated that T cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/ LFA-1 with lifitegrast reduced colitis severity and decreased T cell infiltration in the myenteric plexus. ConclusionsOur present work argues for a role of Glia-T cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1, and suggests that deciphering the functional consequences of Glia-T cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn’s disease.

Bradykinin produced during Plasmodium falciparum erythrocytic cycle drives monocyte adhesion to human brain microvascular endothelial cells

Cerebral malaria (CM) pathogenesis is described as a multistep mechanism. In this context, monocytes have been implicated in CM pathogenesis by increasing the sequestration of infected red blood cells to the brain microvasculature. In disease, endothelial activation is followed by reduced monocyte rolling and increased adhesion. Nowadays, an important challenge is to identify potential pro-inflammatory stimuli that can modulate monocytes behavior. Our group have demonstrated that bradykinin (BK), a pro-inflammatory peptide involved in CM, is generated during the erythrocytic cycle of P. falciparum and is detected in culture supernatant (conditioned medium). Herein we investigated the role of BK in the adhesion of monocytes to endothelial cells of blood brain barrier (BBB). To address this issue human monocytic cell line (THP-1) and human brain microvascular endothelial cells (hBMECs) were used. It was observed that 20% conditioned medium from P. falciparum infected erythrocytes (Pf-iRBC sup) increased the adhesion of THP-1 cells to hBMECs. This effect was mediated by BK through the activation of B2 and B1 receptors and involves the increase in ICAM-1 expression in THP-1 cells. Additionally, it was observed that angiotensin-converting enzyme (ACE) inhibitor, captopril, enhanced the effect of both BK and Pf-iRBC sup on THP-1 adhesion. Together these data show that BK, generated during the erythrocytic cycle of P. falciparum, could play an important role in adhesion of monocytes in endothelial cells lining the BBB.

Diastolic dysfunction is associated with cardiac small vessel disease in ovariectomized females but not in males

Abstract Introduction Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature has still been poorly considered and investigated in animal models of HFpEF. Objective To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction. Method We assessed cardiac function and characterized the coronary microvasculature in two mouse models of HFpEF: mice fed with a high fat diet (HFD) + L-NAME regimen and Leptin receptor deficient (Leprdb/db) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dimorphisms. Results Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Leprdb/db mice, both Male and non OVX female mice develop diastolic dysfunction. Notably female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Leprdb/db mice. Conclusion: Diastolic dysfunction is not always associated with cardiac small vessel disease since Leprdb/db males and C57BL/6J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.

Establishment of an in vivo murine model of immune checkPoint inhibitors cardiotoxicity: emerging role of vascular permeability in pembrolizumab-induced cardiotoxicity

Abstract Introduction Advances in oncology have led to the discovery of immune checkpoint inhibitors (ICIs) that exhibit remarkable results in cancer patients’ survival and tumour regression. Despite their antitumor potential, some life-threatening immune-related adverse effects (AEs) including cardiotoxicity have been reported, while the pathomechanism of the cardiac AEs is obscure. Purpose We herein investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1) in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes in vitro and established the mechanism and progression of Pem’s cardiotoxicity in an in vivo murine model. Methods Primary mAVCs and spleenocytes were isolated and incubated with IPI, PEM and AVE (0-100 μg/ml) for 24h. Cell viability was assessed by MTT assay. Subsequently conditioned media from spleenocytes treated with the aforementioned ICIs was transferred onto the mAVCs for 24h. The experiments were repeated for molecular analyses. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were biotechnologically produced and PEM binding was confirmed by circular dichroism (CD). C57BL6/J male mice were randomized into i. IgG4 and ii. Pem groups and treated for 2 (n = 5/group) and 5 weeks (n = 9/group). IgG4 and Pem were administered intraperitoneally once weekly at 2mg/ml. Pem dose was directly translated from humans. Mice underwent weekly echocardiography analysis, while at the endpoint, mice were sacrificed for myocardial sampling and histology analyses. Results IPI, PEM and AVE did not induce direct cytotoxicity on primary mAVCs, whereas IPI was excluded from the study due to unexpected spleenocyte cytotoxicity. Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce Immune cell (IC)-dependent cytotoxicity at 50 and 100 μg/ml and establishment of inflammation and autophagy in vitro. CD experiments showed that PEM binds on the murine PD-1 ED, which supports for the first time the use of the murine model for the extraction of translational results. In vivo Pem led to %Fractional shortening (FS%) decline, which was significantly reduced versus baseline at 2 weeks and further depressed at 5 weeks. At two weeks, Pem increased ICAM-1 expression as well as iNOS and beclin-1 expression in the myocardium, which was accompanied by intra-cardiac IC infiltration. At five weeks, Pem further increased vascular permeability, as shown by e-selectin and ICAM-1 upregulation and led to increase of cardiac autophagy and inflammation markers. Conclusions Herein we established for the first time a murine in vivo model of PEM cardiotoxicity. PEM induces an IC-mediated mAVCs cytotoxicity, via induction of inflammation, autophagy and ER-stress. Increased vascular permeability and dysregulated endothelial homeostasis seem to orchestrate early-on Pem’s cardiotoxicity manifestation in vivo.

Activation of CB2R by synthetic CB2R agonist, PM289, improves brain endothelial barrier properties, decreases inflammatory response and enhances endothelial repair.

The Cannabinoid 2 Receptor (CB2R) has been found to provide immunological modulation in different cell types. More recently, detection of CB2R in the cerebral endothelium suggests a possible role in the resolution of inflammation at the level of the blood-brain-barrier (BBB). Here, the notion that CB2R upregulation in brain endothelial cells could be exploited to promote vascular protection and BBB integrity was evaluated. Targeting and activation of CB2R was accomplished by a novel and highly specific chromenopyrazole based CB2R agonist, PM289. This study demonstrates that CB2R upregulation is induced as early as 8 h in the cortical vasculature in an experimental mouse model of TBI. Unlike CB2R, CB1R was marginally detected and not significantly induced. In the human brain endothelial cell line, hCMEC/D3 cells, similar induction of CB2R was observed upon stimulation with TNFα. Analysis of transendothelial electrical resistance shows that PM289 markedly prevented the barrier-leakiness induced by TNFα. The BBB is also responsible for maintaining an immunological barrier. The five-fold increase in ICAM1 expression in stimulated endothelial cells was significantly diminished due to CB2R activation. Utilizing wounding assays, results showed that wound repair could be accomplished in nearly half the time when the novel CB2R agonist is present compared to the untreated control. Lastly, mechanistically, the effects of CB2R may be explained by the observed inhibition of the p65 NFκB subunit. Overall, these studies support the notion that targeting and activating CB2R in the brain vasculature could aid in BBB and vascular protection in the context of neuroinflammation.

Abstract P3107: Sexual Dimorphism In Diastolic Dysfunction And Cardiac Small Vessel Disease In Heart Failure With Preserved Ejection Fraction Mouse Models

Introduction: Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature has still been poorly investigated in animal models of HFpEF and sex differences are not always considered. Objective: To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction in two HFpEF mouse models. Method: We assessed cardiac function and characterized the coronary microvasculature in mice fed with a high fat diet (HFD) + L-NAME regimen and in Leptin receptor deficient ( Lepr db/db ) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dimorphisms. Results: Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Lepr db/db mice, both Male and non OVX female mice develop diastolic dysfunction. Notably female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Lepr db/db mice. Conclusion: Diastolic dysfunction is not always associated with cardiac small vessel disease since Lepr db/db males and C57BL/6J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.

#5730 PERLECAN AS A POTENTIAL BIOMARKER OF CARDIOVASCULAR RISK: ROLE OF ENDOTHELIAL GLYCOCALYX IN CHRONIC KIDNEY DISEASE

Abstract Background and Aims Chronic kidney disease (CKD) is associated with cardiovascular disease (CVD). CVD is in turn related to endothelial dysfunction, endothelial dysfunction, and degradation of the endothelial glycocalyx (EG), releasing its components into the bloodstream. The EG consists of glycosaminoglycans and proteoglycans, such as Perlecan. The aim of the study is to analyse the levels of perlecan in plasma in different stages of CKD, and to relate them to age and inflammatory monocytes, as well as their adhesion capacity (CD54/ICAM-1). Method An observational cross-sectional study was carried out. 56 patients were included: advanced chronic kidney disease (ACKD) (n=13), haemodialysis (HD) (n=13), peritoneal dialysis (PD) (n=15) and transplant recipients (TX) (n=15). Thirteen healthy subjects (CT) were analysed. Plasma perlecan levels were quantified using ELISA and phenotyping of monocyte subpopulations (classical (CD14++CD16-), intermediate (CD14+CD16+) and non-classical (CD14+CD16++)), as well as CD54 (ICAM-1) expression by flow cytometry. Statistical analysis: ANOVA and Spearman correlation. Results Patients with CKD had higher plasma levels of perlecan than healthy participants (p-value = 0.044 vs ACKD, 0.028 vs HD and 0.002 vs PD) (Fig. 1). These levels were associated with higher percentage of classical monocytes (p=0,011) intermediate monocytes (p=0,009) and non-classical monocytes (p=0,003) expressing ICAM-1 but not with a higher expression of ICAM-1 per monocyte. Perlecan levels correlate negatively with age (p=0,03) (Table 1). Conclusion Patients with ACKD, HD and PD have elevated plasma perlecan levels compared to CT and TX. Elevated perlecan concentrations are associated with increased ICAM-1 expression on monocytes, which is important for its possible role in the development of atherosclerosis. Perlecan may be postulated as a molecule of interest to assess endothelial and cardiovascular damage in CKD patients.

Diastolic dysfunction is associated with cardiac small vessel disease in ovariectomized females but not in males

Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature have still been poorly considered and investigated in animal models of HFpEF. To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction. We assessed cardiac function and characterized the coronary microvasculature in two mouse models of HFpEF: mice fed with a high fat diet (HFD) + L-NAME regimen and Leptin receptor deficient (Leprdb/db) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dysmorphism. Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Leprdb/db mice, both male and non OVX female mice develop diastolic dysfunction. Notably, female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Leprdb/db mice. Diastolic dysfunction is not always associated with cardiac small vessel disease since Leprdb/db males and C57BL/6 J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.

ICAM1 expression by the microvasculature impairs capillary perfusion which compromises hind limb ischemia recovery in diabetic mice

Chronic limb threatening ischemia (CLTI), which is one of the major complications of diabetes, is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. However, the pathophysiology of this disease is poorly understood and therapeutic targets are missing. Our objective is to explore the contribution of endothelial dysfunction in the development of CLTI in diabetic mice. Hind limb ischemia (HLI) was induced by ligation and resection of the femoral artery in C57BL6/J male mice, in which insulin resistance was induced by a high fat diet, and hyperglycaemia was induced one month later by low dose streptozotocin administrations. According to our previous investigations, ischemic foot re-perfusion, assessed via laser doppler perfusion imaging, was significantly reduced in diabetic animal 28 days after HLI surgery was performed (ratio blood flow in the ischemic leg vs non ischemic leg = 0.27 ± 0.09 vs 0.44 ± 0.21 in control mice; P = 0.03), even though angiogenesis was identical in both groups. On the contrary, we found that impaired ischemic foot re-perfusion was associated with an increased endothelial cell activation attested by an increased ICAM1 expression (P < 0.0001). We then hypothesized that ICAM1, by interacting with white blood cells (WBC), may compromise the perfusion of capillaries with a diameter smaller than a WBC. Accordingly, we found that WBC circulation velocity in the microvasculature was significantly diminished in diabetic mice (533 ± 169 μm/s vs 960 ± 188 in control mice; P = 0.004) and associated with a decreased percentage of capillaries perfusion by BS-1 lectin (78.5 ± 10.5% vs 96.1 ± 5.7 in control mice; P = 0.03). With the aim to test whether ICAM1 overexpression may be responsible for impaired ischemic foot re-perfusion, diabetic mice were administered with anti-ICAM1 antibodies or isotype control for 14 days, starting 14 days after HLI surgery was performed. We found that anti-ICAM1 therapy significantly increased WBC circulation velocity within the microvasculature (779 ± 113 μm/s vs 447 ± 107 in mice treated with isotype control; P < 0.0001) and the percentage of perfused capillary (P = 0.009). Altogether, our results demonstrate that ICAM1 overexpression may compromise hind limb ischemia recovery in diabetic mice by decreasing WBC circulation velocity and impairing capillary perfusion.

Liposome-Encapsulated Eribulin Shows Enhanced Antitumor Activity over Eribulin for Combination Therapy with Anti-PD-1 Antibody.

Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti-programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti-PD-1 Ab was examined in a P-glycoprotein-knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti-PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti-PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti-PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Dengue fever ophthalmic manifestations: A review and update.

Dengue fever, the most common arbovirus disease, affects an estimated 390 million people annually. Dengue virus (DENV) is an RNA virus of the Flaviviridae family with four different serotypes. Dengue haemorrhagic fever is the deadliest form of dengue infection and is characterised by thrombocytopaenia, hypotension, and the possibility of multi-system organ failure. The mechanism hypothesised for DENV viral replication is intrinsic antibody-dependent enhancement, which refers to Fcγ receptor-mediated viral amplification. This hypothesis suggests that the internalisation of DENV through the Fcγ receptor inhibits antiviral genes by suppressing type-1 interferon-mediated antiviral responses. DENV NS1 antibodies can promote the release of various inflammatory mediators in the nuclear transcription factor pathway (NF-κB-dependent), including monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, and IL-8. As a result, MCP-1 increases ICAM-1 expression and facilitates leukocyte transmigration. In addition, anti-DENV NS1 antibodies induce endothelial cell apoptosis via a nitric oxide-regulated pathway. A chain reaction involving pre-existing DENV heterotypic antibodies and innate immune cells causes dysfunction in complement system activity and contributes to the action of autoantibodies and anti-endothelial cells, resulting in endothelial cell dysfunction, blood-retinal barrier breakdown, haemorrhage, and plasma leakage. A spectrum of ocular diseases associated with DENV infection, ranging from haemorrhagic to inflammatory manifestations, has been reported in the literature. Although rare, ophthalmic manifestations can occur in both the anterior and posterior segments and are usually associated with thrombocytopenia. The most common ocular complication is haemorrhage. However, ophthalmic complications, such as anterior uveitis and vasculitis, suggest an immune-mediated pathogenesis.

Single-Cell Multi-Omics Reveal Longitudinal Dynamics of Clonal Architecture and Microenvironment Interactions in Relapsed-Refractory Myeloma

Introduction: Subclonal dynamics in multiple myeloma (MM) have been demonstrated to be strongly influenced by treatment. Moreover, the bone marrow (BM) microenvironment (BME) plays an important role in mediating treatment-associated drug resistance. However, the underlying transcriptional and epigenetic changes across genetically distinct subclones and their interaction with the BME remain poorly characterized. Methods: Single-cell and whole genome sequencing (WGS) was performed in 15 relapsed/refractory MM patients with samples collected prior to the respective salvage therapy (T1) and at the time of subsequent relapse (T2). Using 10X Genomics scATAC- and scRNA-seq protocols, we analysed 44,637 and 37,280 BM CD138-enriched plasma cells after quality control with ArchR and Seurat, respectively. Copy number aberrations (CNAs) were called with inferCNV (scRNA-seq), an approach by Lareau and co-workers (scATAC-seq) and ACESeq (WGS). For mitochondrial DNA (mtDNA) mutations mgatk was used. Cell-cell interactions between subclones and the BME were predicted using CellChat. Results: We developed a WGS-guided clustering strategy to identify individual CNA-based subclones in scRNA- and scATAC-seqdata. Next, this subclone definition was further refined by integrating mtDNA mutations. We found unique mtDNA mutations in 23/53 (43%) CNA-defined subclones. These mutations allowed us to discriminate between subclones with identical CNA profiles or to assign subclones based on CNAs that were otherwise below the WGS detection limit. Furthermore, some mtDNA mutations were jointly enriched in multiple subclones, defining subclonal branches. With our integrative analysis of CNAs and mtDNA mutations, we detected on average 4 (range 1-11) subclones per patient with very similar proportions between the two sc modalities (correlation ρ = 0.97). This allowed us to map the transcriptional profile of each subclone to its epigenetic profile. In patients with multiple subclones and a stable subclonal composition between T1 and T2, we found that subclones similarly adapted their transcriptomic and epigenomic profiles concordantly to the respective treatment. For instance, in a carfilzomib-treated patient, >85% of gene expression changes were shared between both subclones including several heat shock proteins, as well as TNFa signaling via NFkB, apoptosis, hypoxia and the P53 pathway. On the epigenomic level, the transcription factor (TF) motifs of ZNF384 and MEF2 family members were upregulated in both subclones at T2. Looking at patients with subclonal changes between T1 and T2 (n=7), we found a patient in which a subclone carrying bi-allelic TP53 inactivation was depleted by MCL-1 inhibitor treatment. But in a validation experiment, AMO-1 wild-type cell lines were significantly more sensitive to MCL-1 inhibition than bi-allelic TP53 altered AMO-1 cell lines (TP53 del/mut (R175H), p<0.05). However, several surface markers such as CD44 were differentially expressed between the competing subclones. Therefore, we inferred cellular interactions between the respective subclones and BME cells. We identified a strong interaction between CD44 and LGALS9 on monocytes and dendritic cells that was specific for the TP53-inactivated subclone. Extending the interaction analysis to all patients with matched scRNA-seq of the BME (n=9), an average of 32 ligand-receptor MM-BME interactions were predicted per patient. Interestingly, 19% (range 2-12) were subclone specific and primarily driven by Visfatin, ICAM, BAFF, CD23 and Galectin pathways. In addition, we observed shared longitudinal changes in expression of surface markers, including increased ICAM1 expression as well as chromatin co-accessibility at the ICAM1 promotor in the majority of patients. Lastly, known TFs of ICAM1, including IRF1/4, and STAT1/2 demonstrated higher motif activity at T2. Conclusion: Overall, we demonstrate the power of paired bulk and single-cell multi-omics approaches for deciphering changes in the subclonal architecture and cell-cell interactions in the MM ecosystem. Treatment can induce concordant transcriptional and epigenetic changes even in genetically distinct subclones, generating a repertoire of shared targets, including ICAM1. Furthermore, our analysis shows that subclones can interact differently with their BME, which could be one explanation for differential treatment response.

Pioglitazone Attenuates the Effects of Peripheral Inflammation in a Human In Vitro Blood–Brain Barrier Model

Biological mediators secreted during peripheral chronic inflammation reach the bloodstream and may damage the blood-brain barrier (BBB), triggering central nervous system (CNS) disorders. Full-fledged human BBB models are efficient tools to investigate pharmacological pathways and mechanisms of injury at the BBB. We here employed a human in vitro BBB model to investigate the effects of either plasma from inflammatory bowel disease (IBD) patients or tumor necrosis factor α (TNFα), a cytokine commonly released in periphery during IBD, and the anti-inflammatory role of pioglitazone, a peroxisome proliferator-activated receptor γ agonist (PPARγ). The BBB model was treated with either 10% plasma from healthy and IBD donors or 5 ng/mL TNFα, following treatment with 10 µM pioglitazone. Patient plasma did not alter BBB parameters, but TNFα levels in plasma from all donors were associated with varying expression of claudin-5, claudin-3 and ICAM-1. TNFα treatment increased BBB permeability, claudin-5 disarrangement, VCAM-1 and ICAM-1 expression, MCP1 secretion and monocyte transmigration. These effects were attenuated by pioglitazone. Plasma from IBD patients, which evoked higher BBB permeability, also increased ICAM-1 expression, this effect being reversed by pioglitazone. Our findings evidence how pioglitazone controls periphery-elicited BBB inflammation and supports its repurposing for prevention/treating of such inflammatory conditions.

Altered blood-brain barrier transport of nanotherapeutics in lysosomal storage diseases.

Treatment of neurological lysosomal storage disorders (LSDs) are limited because of impermeability of the blood-brain barrier (BBB) to macromolecules. Nanoformulations targeting BBB transcytosis are being explored, but the status of these routes in LSDs is unknown. We studied nanocarriers (NCs) targeted to the transferrin receptor (TfR), ganglioside GM1 or ICAM1, associated to the clathrin, caveolar or cell adhesion molecule (CAM) routes, respectively. We used brain endothelial cells and mouse models of acid sphingomyelinase-deficient Niemann Pick disease (NPD), and postmortem LSD patients' brains, all compared to respective controls. NC transcytosis across brain endothelial cells and brain distribution in mice were affected, yet through different mechanisms. Reduced TfR and clathrin expression were found, along with decreased transcytosis in cells and mouse brain distribution. Caveolin-1 expression and GM1 transcytosis were also reduced, yet increased GM1 levels seemed to compensate, providing similar NC brain distribution in NPD vs. control mice. A tendency to lower NHE-1 levels was seen, but highly increased ICAM1 expression in cells and human brains correlated with increased transcytosis and brain distribution in mice. Thus, transcytosis-related alterations in NPD and likely other LSDs may impact therapeutic access to the brain, illustrating the need for these mechanistic studies.

Radiation enhances the efficacy of EGFR-targeted CAR-T cells against triple-negative breast cancer by activating NF-κB/Icam1 signaling

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with limited treatment options. Epidermal growth factor receptor (EGFR) is reported to be expressed in 50%-75% of TNBC patients, making it a promising target for cancer treatment. Here we show that EGFR-targeted chimeric antigen receptor (CAR) Tcell therapy combined with radiotherapy provides enhanced antitumor efficacy in immunocompetent and immunodeficient orthotopic TNBC mice. Intriguingly, this combination therapy resulted in a substantial increase in the number of tumor-infiltrating CAR-T cells. The efficacy of this combination was independent of tumor radiosensitivity and lymphodepleting preconditioning. Cytokine profiling showed that this combination did not increase the risk of cytokine release syndrome (CRS). RNA sequencing (RNA-seq) analysis revealed that EGFR-targeting CAR-T therapy combined with radiotherapy increased the infiltration of CD8+ T and natural killer (NK) cells into tumors. Mechanistically, radiation significantly increased Icam1 expression on TNBC cells via activating nuclear factor κB (NF-κB) signaling, thereby promoting CAR-T cell infiltration and killing. These results suggest that CAR-T therapy combined with radiotherapy may be a promising strategy for TNBC treatment.

An endothelial proinflammatory phenotype precedes the development of the engraftment syndrome after autologous Hct.

Engraftment syndrome (ES) is a common complication after autologous hematopoietic cell transplantation (auto-HCT) whose pathophysiological substrate remains unclear. We investigated whether endothelial damage could contribute to ES. Circulating ECs-damage biomarkers were measured in plasma from patients with (ES; n = 14) or without ES (non-ES; n = 20), collected at different time points: before HCT, 5 (S5) and 10 days (S10) after HCT, and at either the ES onset (SON) or the discharge day (SDIS). Also, cultured endothelial cells (ECs) were exposed to serum samples, obtained at the same points, to evaluate changes in ECs-activation (ICAM-1, VE-Cadherin) biomarkers, the reactivity of ECs towards leukocytes, and activation of intracellular signaling proteins related to inflammation (p38MAPK) and proliferation (Erk1/2). Results showed that circulating VWF, sTNFR1 and sVCAM-1 levels were higher in ES patients at all the points assessed, especially at SON. In vitro results showed an increased ICAM-1 expression on ECs exposed to ES samples vs. non-ES samples, especially to S5, with elevated leukocyte adhesion. Also, a lower VE-Cadherin expression and an increased phosphorylation of p38MAPK and Erk1/2 proteins were observed in ECs exposed to ES vs. non-ES samples. Our results indicate that endothelial activation precedes ES development and could be one of its pathophysiological substrates.

Comparative study of the effects of cigarette smoke versus next generation tobacco and nicotine product extracts on endothelial function

Tobacco smoking and hemodynamic forces are key stimuli for the development of endothelial dysfunction. As an alternative to smoking, next generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their potential pro-inflammatory and atherogenic effects on the endothelium. In this study, we analyzed key parameters of endothelial function after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine. All experiments were performed under atheroprotective high laminar or atherogenic low flow with primary human endothelial cells.Treatment with 3R4F, but not alternative smoking products, reduced endothelial cell viability and wound healing capability via the PI3K/AKT/eNOS(NOS3) pathway. Laminar flow delayed detrimental effects on cell viability by 3R4F treatment. 3R4F stimulation led to activation of NRF2 antioxidant defense system at nicotine concentrations ≥0.56 μg/ml and increased expression of its target genes HMOX1 and NQO1. Treatment with HTP revealed an induction of HMOX1 and NQO1 at dosages with ≥1.68 μg/ml nicotine, whereas e-cig and nicotine exposure had no impact. Analyses of pro-inflammatory genes revealed an increased ICAM1 expression under 3R4F treatment. 3R4F reduced VCAM1 expression in a dose-dependent manner; HTP treatment had similar but milder effects; e-cig and nicotine treatment had no impact. SELE expression was induced by 3R4F under static conditions. High laminar flow prevented this upregulation. Stimulation with laminar flow led to downregulation of CCL2 (MCP-1). From this downregulated level, only 3R4F increased CCL2 expression at higher concentrations. Finally, under static conditions, all components increased adhesion of monocytes to endothelial cells. Interestingly, only stimulation with 3R4F revealed increased monocyte adhesion under atherosclerosis-prone low flow.In conclusion, all product categories activated anti-oxidative or pro-inflammatory patterns. NGP responses were typically lower than in 3R4F exposed cells. Also, 3R4F stimulation led to an impaired endothelial wound healing and induced a pro-inflammatory phenotype compared to NGP treatment.

Transforming growth factor (TGF)-β1 and interferon (IFN)-γ differentially regulate ICAM-1 expression and adhesion of Toxoplasma gondii to human trophoblast (BeWo) and uterine cervical (HeLa) cells

Toxoplasma gondii is a parasite able to infect various cell types, including trophoblast cells. Studies have demonstrated that interleukin (IL)-10, transforming growth factor (TGF)-β1 and interferon (IFN)-γ are involved in the susceptibility of BeWo trophoblast cells to T. gondii infection. Furthermore, T. gondii is able to adhere to the plasma membrane of host cells through intercellular adhesion molecule (ICAM)-1. Thus, the present study aimed to assess the role of IL-10, TGF-β1 and IFN-γ in the expression of ICAM-1 in BeWo and HeLa cells and to analyze the role of ICAM-1 in the adhesion and invasion of T. gondii to these cells under the influence of these cytokines. For this purpose, BeWo and HeLa cells were treated or not, before and after T. gondii infection, with rIL-10, rTGF-β1 or rIFN-γ. For the BeWo cells, rIL-10 and rTGF-β1 favored susceptibility to infection, but only rTGF-β1 and rIFN-γ increased ICAM-1 expression, and TNF-α release. On the other hand, rIFN-γ downregulated the expression of ICAM-1 triggered by T. gondii in HeLa cells, leading to control of the infection. Moreover, we observed that upregulation of ICAM-1, mediated by cytokine's stimulation, in BeWo and HeLa cells resulted in a high number rate of both parasite adhesion and invasion to these cells, which were strongly reduced after ICAM-1 neutralization. Likewise, the blockage of ICAM-1 molecule also impaired T. gondii infection in human villous explants. Taken together, these findings demonstrate that TGF-β1 and IFN-γ differentially regulate ICAM-1 expression, which may interfere in the adhesion/invasion of T. gondii to BeWo and HeLa cells for modulating susceptibility to infection.

Melatonin, a Potential Therapeutic Agent for Preeclampsia, Reduces the Extrusion of Toxic Extracellular Vesicles from Preeclamptic Placentae.

Preeclampsia, characterised by maternal endothelial cell activation, is triggered by toxic factors, such as placental extracellular vesicles (EVs) from a dysfunctional placenta. The increased oxidative stress seen in the preeclamptic placenta links to endoplasmic reticulum (ER) stress. The ER regulates protein folding and trafficking. When the ER is stressed, proteins are misfolded, and misfolded proteins are toxic. Misfolded proteins can be exported from cells, via EVs which target to other cells where the misfolded proteins may also be toxic. Melatonin is a hormone and antioxidant produced by the pineal gland and placenta. Levels of melatonin are reduced in preeclampsia. In this study we investigated whether melatonin treatment can change the nature of placental EVs that are released from a preeclamptic placenta. EVs were collected from preeclamptic (n = 6) and normotensive (n = 6) placental explants cultured in the presence or absence of melatonin for 18 h. Misfolded proteins were measured using a fluorescent compound, Thioflavin-T (ThT). Endothelial cells were exposed to placental EVs overnight. Endothelial cell activation was measured by the quantification of cell-surface ICAM-1 using a cell-based ELISA. EVs from preeclamptic placentae carried significantly (p < 0.001) more misfolded proteins than normotensive controls. Incubating preeclamptic placental explants in the presence of melatonin (1 µM and 10 µM) significantly (p < 0.001) reduced the misfolded proteins carried by EVs. Culturing endothelial cells in the presence of preeclamptic EVs significantly increased the expression of ICAM-1. This increased ICAM-1 expression was significantly reduced when the endothelial cells were exposed to preeclamptic EVs cultured in the presence of melatonin. This study demonstrates that melatonin reduces the amount of misfolded proteins carried by EVs from preeclamptic placentae and reduces the ability of these EVs to activate endothelial cells. Our study provides further preclinical support for the use of melatonin as a treatment for preeclampsia.

AIM2 deletion enhances blood-brain barrier integrity in experimental ischemic stroke.

AimsIschemic stroke is a life‐threatening disease with limited therapeutic strategies. Blood‐brain barrier (BBB) disruption is a critical pathological process that contributes to poor outcomes in ischemic stroke. We previously showed that the microglial inhibition of the inflammasome sensor absent in melanoma 2 (AIM2) suppressed the inflammatory response and protected against ischemic stroke. However, whether AIM2 is involved in BBB disruption during cerebral ischemia is unknown.MethodsMiddle cerebral artery occlusion (MCAO) and oxygen‐glucose deprivation/reoxygenation (OGD/R) were used to mimic cerebral ischemia in mice and brain microvascular endothelial cells (HBMECs), respectively. The infarct volume, neurological deficits, and BBB permeability were measured in mice after MCAO. Transendothelial electrical resistance (TEER) and neutrophil adhesion to the HBMEC monolayer were assessed after OGD/R treatment. Western blot and immunofluorescence analyses were conducted to evaluate the expression of related proteins.ResultsAIM2 was shown to be expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. AIM2 deletion reduced the infarct volume, improved neurological and motor functions, and decreased BBB disruption. In vitro, OGD/R significantly increased the protein levels of AIM2 and ICAM‐1 and decreased those of the tight junction (TJ) proteins ZO‐1 and occludin. AIM2 knockdown effectively protected BBB integrity by promoting the expression of TJ proteins and decreasing ICAM‐1 expression and neutrophil adhesion. Mechanistically, AIM2 knockdown reversed the OGD/R‐induced increases in ICAM‐1 expression and STAT3 phosphorylation in brain endothelial cells. Furthermore, treatment with the p‐STAT3 inhibitor AG490 mitigated the effect of AIM2 on BBB breakdown.ConclusionOur findings indicated that inhibiting AIM2 preserved the BBB integrity after ischemic stroke, at least partially by modulating STAT3 activation and that AIM2 may be a promising therapeutic target for cerebral ischemic stroke.