Primary Human Endothelial Cells Research Articles

Abstract B024: KO-2806, a next-generation farnesyltransferase inhibitor, potentiates the antitumor activity of cabozantinib in clear cell renal cell carcinoma models

Abstract Conventional treatments for advanced clear cell renal cell carcinoma (ccRCC) include tyrosine kinase inhibitors (TKI) that target angiogenesis, a critical element for ccRCC tumor growth and survival. Cabozantinib, a TKI that inhibits VEGFR, AXL, and MET, is an approved therapy in advanced RCC. However, cabozantinib has limited therapeutic benefit; for example, patients with prior VEGFR TKI therapy had an overall response rate of 21% and median progression-free survival of 7.4 months. Thus, there is an urgent need to identify combination drug partners that can improve the depth and/or durability of clinical responses to cabozantinib. Here, we present preclinical evidence that supports combining the farnesyltransferase inhibitor (FTI), KO-2806, with cabozantinib in ccRCC. KO-2806 is a next-generation farnesyltransferase inhibitor with increased potency and improved pharmacokinetic properties relative to earlier FTI drug candidates and is poised to enter a first-in-human clinical trial. We demonstrate that addition of KO-2806 enhanced tumor growth inhibition of cabozantinib in multiple ccRCC cell line- and patient-derived xenograft models, resulting in either tumor stasis or regression. Immunohistochemical analysis of angiogenic markers in 786-O tumors revealed that, consistent with its mechanism of action, cabozantinib significantly reduced angiogenesis, an effect that was deepened by KO-2806. In support of this, cell viability experiments performed in primary human endothelial cells showed that the combination of KO-2806 and cabozantinib was more effective in reducing cell viability than cabozantinib alone. In addition, we investigated the mechanism underlying the improved response of tumors with the KO-2806-cabozantinib combination by analyzing the major growth signaling pathways in 786-O tumors. Cabozantinib alone reduced both MAPK and PI3K/AKT/mTOR signaling, but addition of KO-2806 resulted in enhanced inhibition of mTOR signaling and increased cell cycle arrest compared to either agent alone. We hypothesize that exposure to anti-angiogenic TKIs render ccRCC tumors more hypoxic, and thus more reliant on compensatory, oncogenic mechanisms like mTOR signaling that allow them to adapt to a nutrient- and oxygen-deprived environment. Mechanistically, KO-2806 potently inhibits the farnesylation, and hence the activity, of the farnesylation-dependent protein RHEB, a positive regulator of mTOR, suggesting that the improved anti-tumor efficacy of the combination may arise through RHEB inhibition in this model. Further in vitro and in vivo studies are underway to establish the scope and mechanistic underpinnings of the effects of the KO-2806-cabozantinib combination and strengthen the therapeutic rationale for the investigation of this approach in the treatment of patients with ccRCC. Citation Format: Jovylyn Gatchalian, Linda Kessler, Hetika Patel, Stacia Chan, Francis Burrows, Shivani Malik. KO-2806, a next-generation farnesyltransferase inhibitor, potentiates the antitumor activity of cabozantinib in clear cell renal cell carcinoma models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B024.

P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2

Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Evaluation of the impact of tragacanth/xanthan gum interpolymer complexation with chitosan on pharmaceutical performance of gels with secnidazole as potential periodontal treatment

Periodontitis consists a group of dental disorders that affect about 70 % of the world population. The therapy mainly relies on mechanical removing bacterial biofilm, nevertheless, local or systemic antibacterial agents play a key role in treating the acute conditions. Secnidazole is a newer derivative of commonly used metronidazole with high safety profile and broad spectrum of antimicrobial activity. The aim of the study was to evaluate the applicability of polyelectrolyte complex-based hydrogels composed of anionic tragacanth with addition of xanthan gum and cationic chitosan as carriers for buccal/intra pocket delivery of secnidazole. Prepared hydrogels with 5 % and 10 % (w/w) drug content were evaluated pharmaceutically towards inter alia physicomechanical, rheological and thermal properties, drug release kinetics, swelling behavior or antimicrobial activity. Cytotoxicity against human primary umbilical vein endothelial cells was also assessed with two independent method. Stable compositions with secnidazole were obtained, however, various miscibility of the drug with the polymers was noted. By adding chitosan, antibacterial activity and swelling performance of the gels were improved, nevertheless, drop of the mucoadhesiveness was also recorded. Hydrogels with 5 % secnidazole were selected as effective antimicrobial compositions with the highest cytocompatibility. They might be considered as promising for oromucosal application with special attention given to SEC as an alternative locally administered antimicrobial agent.

Hyperglycolysis in endothelial cells drives endothelial injury and microvascular alterations in peritoneal dialysis.

Endothelial cell (EC) dysfunction leading to microvascular alterations is a hallmark of technique failure in peritoneal dialysis (PD). However, the mechanisms underlying EC dysfunction in PD are poorly defined. We combined RNA sequencing with metabolite set analysis to characterize the metabolic profile of peritoneal ECs from a mouse model of PD. This was combined with EC-selective blockade of glycolysis by genetic or pharmacological inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in vivo and in vitro. We also investigated the association between peritoneal EC glycolysis and microvascular alterations in human peritoneal samples from patients with end-stage kidney disease (ESKD). In a mouse model of PD, peritoneal ECs had a hyperglycolytic metabolism that shunts intermediates into nucleotide synthesis. Hyperglycolytic mouse peritoneal ECs displayed a unique active phenotype with increased proliferation, permeability and inflammation. The active phenotype of mouse peritoneal ECs can be recapitulated in human umbilical venous ECs and primary human peritoneal ECs by vascular endothelial growth factor that was released from high glucose-treated mesothelial cells. Importantly, reduction of peritoneal EC glycolysis, via endothelial deficiency of the glycolytic activator PFKFB3, inhibited PD fluid-induced increases in peritoneal capillary density, vascular permeability and monocyte extravasation, thereby protecting the peritoneum from the development of structural and functional damages. Mechanistically, endothelial PFKFB3 deficiency induced the protective effects in part by inhibiting cell proliferation, VE-cadherin endocytosis and monocyte-adhesion molecule expression. Pharmacological PFKFB3 blockade induced a similar therapeutic benefit in this PD model. Human peritoneal tissue from patients with ESKD also demonstrated evidence of increased EC PFKFB3 expression associated with microvascular alterations and peritoneal dysfunction. These findings reveal a critical role of glycolysis in ECs in mediating the deterioration of peritoneal function and suggest that strategies targeting glycolysis in peritoneal ECs may be of therapeutic benefit for patients undergoing PD.

Effect of Integrin α 4β 1 -VCAM-1 Interactions in JAK2V617F Myeloproliferative Neoplasm Pro-Thrombotic Interactions

Introduction: The JAK2 V617Fmutation, which occurs in up to 95% of polycythemia vera (PV) myeloproliferative neoplasm (MPN) patients, increases thrombosis risk 6-fold. A recent study in PV mice demonstrated that blocking JAK2 V617F+ leukocyte-mediated β1/β2 integrin interactions with anti-VCAM-1 antibodies decreases thrombosis. Interestingly, hydroxyurea (HU), a common PV treatment, reduces leukocyte and red cell α4β1 integrin expression. Overall, JAK2 V617F+ mouse models show inconsistent thrombosis and bleeding phenotype, hindering pre-clinical study reproducibility; therefore, we have developed an in vitro endothelialized microfluidic model to assess MPN-flow behavior. We hypothesized that in our microfluidic model, disrupting α4β1 integrin expression on JAK2 V617F+ leukocytes would reduce pro-adhesive interactions. We tested this hypothesis by assessing JAK2 V617F+ MPN blood flow in untreated and hydroxyurea treated subjects and through addition of a monoclonal antibody against α4β1 integrin, natalizumab. Methods: Primary human umbilical vein endothelial cells (HUVECs) were exposed to venous shear (15 dynes/cm 2) in microfluidics for a 4-day culture period. On day 4, endothelialized microfluidics were treated +/- 10 ng/mL TNFα for 4 hours. Blood samples were obtained from consented JAK2 V617F MPN individuals aged >18 years with ET, PV, or MF as diagnosed by the WHO 2016 Criteria identified in the M Health Fairview Masonic Cancer Center Clinics. Healthy age, race, and sex-matched blood samples served as controls. Clinical data on patients was collected and stored in REDCAP. Platelets and leukocytes were visualized using Calcein AM. Samples were treated +/- 0.25 μg/mL natalizumab for 30 minutes, then perfused through the microfluidic under a microscope to observe blood-endothelial interactions in real-time. Post-experiment, videos were analyzed to quantify velocity and adhesion of platelets and leukocytes for each treatment condition. Results: When perfused over TNF-α activated endothelium, both controls ( JAK2 negative, n=11) and JAK2 V617F MPN (n=20) had a significant reduction in cell velocity. Furthermore, compared to controls, JAK2 V617F MPN individuals exhibited significantly lower cell velocities. In multi-variate analysis, reduction in cell velocity was independent of white blood cell, hemoglobin, hematocrit or platelet counts. Hydroxyurea is a standard therapy in JAK2 V617F MPN and is known to reduce α4β1 expression. Therefore, we performed subgroup analysis in our JAK2 V617F MPN cohort based on treatment. When perfused over TNF-α activated endothelium, individuals with JAK2 V617F MPN (n=9) not on therapy (newly diagnosed) or on phlebotomy/aspirin only had a significant reduction in cell velocity. In the same conditions, HU-treated JAK2 V617F MPN patients (n=9) had no change in cell velocity. There was no difference in complete blood counts between these groups. In a separate subgroup analysis, compared to untreated JAK2 V617F MPN patients (n=9), interferon-treated JAK2 V617F MPN patients (n=5) had a greater reduction in cell velocity. Last, we specifically targeted α4β1 integrin by adding natalizumab to either control (n=7) or untreated JAK2 V617F MPN (n=3) samples. On inactivated endothelium, treatment of either control or JAK2 V617F MPN blood with natalizumab did not significantly change cell velocity. On a TNF-α activated endothelium, treatment of control (n=7) and JAK2 V617F MPN blood (n=3) with natalizumab led to a recovery in cell velocity. Conclusion: Our data suggests that the interaction between integrin α4β1 on JAK2 V617F cell surface and VCAM-1 on activated endothelium is important for initiation of pro-thrombotic interactions. JAK2 V617F MPN individuals treated with HU exhibit decreased adhesion to activated endothelium compared to control; JAK2 V617F MPN individuals treated with interferon exhibit increased adhesion. In the microfluidic system, based on current findings, blockage of integrin α4β1-VCAM-1 interactions using natalizumab is a potential therapeutic strategy. Further mechanistic studies evaluating interferon and cell populations changed by therapies as they pertain to integrin α4β1 are underway.

Comparison of the Enzymatic and Cellular Profiles of Clinical JAK2 Inhibitors for the Treatment of Myelofibrosis

Introduction: Three Janus kinase 2 (JAK2) inhibitors (JAKinibs) have been approved by the FDA for the treatment of myelofibrosis (MF): ruxolitinib, fedratinib, and pacritinib; a fourth one, momelotinib, is currently under FDA review. In addition to JAK2, these drugs also inhibit other kinases with distinct profiles with potential implications in their observed pharmacology. Published data on kinase inhibitory profiles of the four JAKinibs are limited and were determined under differing assay conditions (Wernig G et al. Cancer Cell 2008; Pardanani A et al. Leukemia 2009; Quintás-Cardama A et al. Blood 2010; Singer J et al. J Exp Pharm 2016), which limits head-to-head comparisons and understanding of their JAK2 inhibition unrelated effects. Here, we present full kinome profiling data for these four JAKinibs. Additionally, we have compared their effects on signaling and proliferation in myeloid cells and other human cells. Methods and Results: Enzymatic assays performed in the presence of 1 mM ATP showed that ruxolitinib is the most potent JAK2 inhibitor (IC 50 4 nM) followed by fedratinib (14 nM), momelotinib (51 nM), and pacritinib (53 nM). Inhibition of other JAK family proteins such as JAK1 and TYK2 were also observed to varying degrees with each drug, and these enzymatic activities were consistent with inhibition of STAT signaling in response to different cytokines in secreted embryonic alkaline phosphatase (SEAP) cellular reporter assays (HEK-Blue Detection, InvivoGen, San Diego, CA). Full kinome analyses (372 activity assays of 356 unique wild-type protein kinases) were conducted (Reaction Biology, Malvern, PA) with determination of percentage inhibition at 100 nM of each drug with 10 μM ATP. Kinases exhibiting >50% inhibition with any of the drugs were then evaluated for dose-response to each drug with 100 μM ATP. The results shown in the Figure demonstrate that fedratinib, momelotinib, and pacritinib inhibited additional kinases outside of the JAK family with similar potency to JAK2 inhibition, whereas ruxolitinib was relatively selective for JAK2 inhibition. In contrast to published reports on the inhibitory activities of pacritinib and momelotinib against ALK2 ( ACVR1), the inhibition of ALK2 observed here was 5-10 times weaker than JAK2 inhibition. Furthermore, pacritinib and momelotinib were both weaker inhibitors of ALK2 activity when compared with their potency towards more than 30 other kinases screened. Fedratinib, pacritinib, and momelotinib, but not ruxolitinib, exhibited high inhibitory potencies against FLT3 and/or KIT kinases, both of which are critical for the self-renewal and differentiation of normal hematopoietic stem cells. In cellular assays monitoring JAK2 signaling (pSTAT5 level in SET2 cells), ruxolitinib was the most potent agent (IC 50 14 nM), followed by momelotinib (205 nM), pacritinib (429 nM), and fedratinib (672 nM). Similar trends were observed in other cellular assays assessing antiproliferative effects in JAK2-dependent myeloid cell lines. In cells not dependent on JAK pathway signaling for growth in culture, such as primary human fibroblasts and endothelial cells, ruxolitinib showed weak or no inhibition at 5 μM, while fedratinib, pacritinib and momelotinib showed anti-proliferative effects with low micromolar IC 50s. Similar trends were observed in human HEK293 and HepG2 cell lines, which are commonly used to assess drug effects on general cell health. We analyzed the effects of the JAKinibs on the differentiation of CD34 + hematopoietic stem and progenitor cells (HSPCs) derived from healthy donors at concentrations observed in the plasma of MF patients enrolled in clinical trials. Consistent with their activity on multiple kinases, including FLT3 and KIT, fedratinib and pacritinib elicited the most pronounced decrease in overall viability, affecting HSPCs and megakaryocytes. In contrast, ruxolitinib as a more selective JAK2 inhibitor, showed the least effect on the differentiation of normal CD34 + HSPCs. Conclusion: JAKinibs clinically evaluated for treatment of MF exhibit distinct kinase inhibition profiles and cellular activities. Amongst the agents tested in this study, ruxolitinib was the most potent and selective JAK2 inhibitor. At concentrations several fold above clinically relevant concentrations, ruxolitinib had no observable effects on the health of cells not reliant on JAK/STAT mediated signaling.

O-GlcNAcylation Inhibition Upregulates Connexin43 Expression in the Endothelium to Protect the Tight Junction Barrier in Diabetic Retinopathy.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on connexin43 (Cx43) expression and its subsequent effects on tight junction properties in diabetic retinopathy (DR). O-GlcNAcylation levels in primary human retinal vascular endothelial cells (HRVECs) and retinas from rats with diabetes were regulated by treatment with Thiamet G or alloxan. Immunoprecipitation was used to examine the relationship between O-GlcNAcylation and Cx43 expression. Stable overexpression and knockdown of Cx43 in HRVECs were achieved using lentivirus constructs; further, their effects on occludin and zonula occluden-1 (ZO-1) expression and tight junction barrier function were determined. O-GlcNAcylation level increased significantly, whereas Cx43 expression decreased in retinas from rats with diabetes and HRVECs cultured under high-glucose conditions. Immunoprecipitation revealed that Cx43 was modified by O-GlcNAcylation and phosphorylation simultaneously. O-GlcNAcylation inhibition negatively regulated both total Cx43 and phosphorylated Cx43 expression, subsequently disrupting tight junction properties. Conversely, Cx43 overexpression reversed the disruption of tight junction properties and downregulated vascular endothelial growth factor expression. Consistently, Cx43 overexpression increased transendothelial electrical resistance values in HRVEC layers. O-GlcNAcylation negatively regulated Cx43 expression, contributing to the disruption of the blood retinal barrier. However, O-GlcNAcylation inhibition and Cx43 overexpression could reverse the tight junction disruption. Therefore, O-GlcNAcylation inhibition is a potential target for avoiding tight junction disruption through the Cx43 pathway in DR.

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2).MethodsIn this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis.ResultsElevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-β upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion.ConclusionsThese findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs 4 Regulates Pulmonary Vascular Hyperpermeability through Destruction of Glycocalyx in Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) has no specific and effective treatment, and there is an urgent need to understand its pathogenesis. Therefore, based on the hypothesis that molecules whose expression is upregulated in injured pulmonary vascular endothelial cells (VECs) are involved in the pathogenesis of ARDS, we conducted a study to elucidate the molecular mechanisms and identify target factors for treatment. Primary human lung microvascular endothelial cells (HMVEC-Ls) were stimulated with lipopolysaccharide (LPS) or poly (I:C) and analyzed via a microarray to identify target genes for ARDS. We found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) was induced in murine lung VECs in an LPS-mediated ARDS model. Elevated ADAMTS4 was also observed by the immunostaining of lung samples from ARDS patients. The suppression of ADAMTS4 by siRNA in VECs ameliorated LPS-stimulated vascular permeability. The impairment of the cell surface expression of syndecan-1, a marker of the glycocalyx that is an extracellular matrix involved in vascular permeability, was dramatically inhibited by ADAMTS4 suppression. In addition, the suppression of ADAMTS4 protected against LPS-induced reductions in syndecan-1 and the adherens junction protein vascular endothelial cadherin. These results suggest that ADAMTS4 regulates VEC permeability in ARDS and may be a predictive marker and therapeutic target for ARDS.

Growth differentiation factor 15 (GDF15) is elevated in human plaques and promotes endothelial-to-mesenchymal transition (EndMT)

Abstract Introduction and Purpose Growth Differentiation Factor 15 (GDF15) is a member of the TGF-β superfamily and is upregulated under conditions of injury and stress such as hypoxia. GDF15 serum concentrations correlate with inflammation, cardiac fibrosis and unfavorable prognosis in cardiovascular diseases. The functional role of GDF15 in endothelial cells is largely unknown. Here, we investigate a possible role of GDF15 in the transdifferentiation of endothelial cells (EC) into mesenchymal cells (EndMT). Methods and Results GDF15 is expressed at mRNA and protein level in primary human ECs and is secreted as determined by qPCR, single cell sequencing (scRNA-seq) and enzyme-linked immunosorbent assay (mean ± SEM, 743.8±39.3 pg/ml, n=3). Analysis of vascular sections from Ldlr-/- mice fed a high-fat diet using spatial transcriptomics revealed increased GDF15 expression. ScRNA-seq analysis of human plaques and normal vessel sections from the same individuals revealed a 22-fold ± 5.48 (p<0.05) increase in GDF15 in ECs located in plaques compared to ECs in control vessel regions. Experiments in cultured human endothelial cells showed increased GDF15 mRNA expression qPCR (+1.7-fold control, p<0.05) and scRNA-seq analysis after EndMT induction. Treatment with increasing levels of recombinant GDF15 (10, 50 and 150 ng/ml) induced EndMT as measured by expression of the EndMT marker calponin (+3.36, 7.99 and 9.53-fold, all p<0.05). Interestingly, EndMT induction decreased extracellular GDF15 levels (-54.6%, p<0.05), whereas intracellular levels in EC were increased (+1.9-fold control, p<0.05). The use of GDF15 neutralising antibodies (1 µg/ml) reduced soluble GDF15 levels by 97.7% as measured by ELISA (p<0.001). Treatment with neutralising antibodies against GDF15 reduced EndMT induction by 54% as measured by the EndMT marker calponin (p<0.05). Conclusion In conclusion, GDF15 is elevated in mouse and human plaques, and in EndMT. Treatment with GDF15 induces EndMT, whereas inhibition by neutralising antibodies reduces EndMT induction. These data are consistent with a functional role of GDF15 in endothelial dysfunction and atherogenesis.

IL-1 alpha is myristoylated for monocyte surface translocation and promotes atherosclerosclerosis in mice

Abstract Background The role of Interleukin-1 alpha (IL-1α) and beta (IL-1β) in the pathogenesis of atherosclerosis in vivo has been controversial for years. In contrast to IL-1β, which is dependent on the NLR family pyrin domain containing 3 (NLRP3) inflammasome, IL-1α can be tethered to the plasma membrane (csIL-1α) where it exerts pro-inflammatory effects. We characterised the role of IL-1α and the Nlrp3 inflammasome in a nongenetic hyperlipidemic mouse model and examined the function of csIL-1α in human monocytes. Methods Atherosclerosis was induced by a single injection of mutant PCSK9-AAV8 virus in wild-type (WT, referred to as PCSK9-AAV), Il1a-/-, Nlrp3-/-, and Il1b-/- mice, in combination with high-fat western diet (21% fat, 0.2% cholesterol) for 12 weeks. Plaque size and lipid content, were histologically analysed. The serum proteome was studied using a targeted multiplex PEA (Olink Proteomics). Human primary monocytes and human umbilical vein endothelial cells (HUVECs) were used to examine csIL-1α-IL1R interaction by analyzing VCAM1 expression and monocyte adhesion. LPS was used to induce IL-1α protein synthesis and membrane translocation in vitro. Myristoylation was measured via flow cytometry. Results Il1a-/- animals showed significantly reduced atherosclerotic lesion size (-62% vs. PCSK9-AAV) and fat accumulation within the plaque (-64% vs. PCSK9-AAV). Knockout of Nlrp3 and IL-1β did not reduce plaque size or lipid content, indicating an Nlrp3-independent mechanism of the protective effect of Il1a-/-. Circulating pro-inflammatory cytokines such as IL-1α, IL-1β, and IL-6 were downregulated in serum of Il1a-/-, Nlrp3-/-, and Il1b-/- mice compared to PCSK9-AAV mice suggesting that the development of atherosclerotic plaques is independent of circulating inflammatory cytokines at an early stage. Therefore, we investigated the role of csIL-1α on human monocytes. Stimulation with LPS showed enrichment of csIL-1α by 13.2% (p<0.05) on monocytes without releasing IL-1α. Treatment of HUVECs with LPS-stimulated monocytes resulted in increased VCAM1 expression (+22.2%, p<0.05) and monocyte adhesion (1.9 fold, p<0.05) through a csIL-1α-IL1R interaction which was abolished by the addition of neutralising antibodies for IL1R1 and IL-1α (p<0.05, vs. LPS-treated monocytes). To investigate whether the IL-1α translocation is mediated by protein myristoylation, human monocytes were pre-incubated with the n-myristoyl-transferase inhibitor IMP-1088 before LPS stimulation, resulting in a reduction of csIL-1α expression (p<0.05, vs. LPS-treated monocytes). Conclusion This study demonstrated a protective effect of Il1a deficiency on the development of atherosclerosis. CsIL-1α mediates monocyte-to-endothelial adhesion by increasing VCAM1 expression through endothelial IL1R1 signaling. The data underscore the importance of the juxtacrine signaling of IL-1α and its role in the development of atherosclerosis independent of circulating cytokine levels.

Histone Mono-methylation by SETD7 Orchestrates Angiogenic Response in Diabetic Limb Ischemia

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose: To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology: Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G, (Figure 1). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle (Figure 2). Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls (Figure 2) Conclusion: Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.

Scalable and stable human pluripotent stem cell-derived microvascular-like endothelial cells for cardiac regeneration and disease modelling applications

Abstract Introduction The microvasculature is a vital constituent of the adult human vasculature 1. Cardiac tissue engineering, in vitro models of cardiac remodelling and regeneration, and disease models of the microvasculature would benefit from the inclusion of well-characterised, phenotypically stable microvascular-like endothelial cells (ECs) that could be generated at scale without xenogeneic reagents. Primary microvascular ECs have been utilised for such applications however, they are susceptible to passaging-induced senescence². Moreover, alternative solutions including current protocols for the derivation of human pluripotent stem cell-derived ECs (hPSC-ECs) generate cells with a heterogenous vascular identity that often transdifferentiate into non-EC lineages³. Purpose The purpose of this study was to create a 3D, xenogeneic-free differentiation protocol that yielded stable hPSC-derived microvascular-like ECs (hPSC-MVECs) at scale. Methods Endothelial differentiation of hPSCs was conducted in 2D adherent monolayers and within stirred 3D bioreactors for 12 days. Thereafter, CD31pos ECs were isolated via fluorescence-activated cell sorting (FACS) and cultured for a further 14 days. Gene expression analysis of hPSCs undergoing 2D and 3D differentiation was evaluated via RT-qPCR. The day 26 hPSC-ECs were treated with VEGFA (0 – 50 ng/ml) for a further 7 days and evaluated for CD31 expression via high-content image analysis. The angiogenic profiles of 2D-, 3D-, and 3D-hPSC-ECs cultured in high concentration VEGFA (herein, 3D+V), relative to primary Human Cardiac Microvascular Endothelial Cells (HMVEC-Cs) was assessed using the Proteome Profiler Human Angiogenesis Array. Transcriptomic analysis of these EC populations was subsequently conducted via single-cell RNA sequencing (scRNA-seq). Results Complex vascular clusters emerged by day 12 of the 3D differentiation protocol (fig. 1). The emergent 3D hPSC-ECs had a comparable expression of PECAM1 and CDH5 relative to 2D-derived hPSC-ECs however, VEGFA expression was significantly reduced (P<0.01, fig. 2A). VEGFA treatment at 50 ng/ml yielded phenotypically stable hPSC-ECs (3D+V, fig. 2B). Principal component analysis of the angiogenic factors assayed for revealed 3D+V hPSC-ECs clustered with HMVEC-Cs (fig. 2C). scRNA-seq identified a subset of 3D-derived hPSC-ECs expressing genes associated with microvascular vessels (GMFG, PLVAP), capillary ECs (RGCC, GLUL), and angiogenic tip cells (ESM1, HSPG2). Conclusion The scalable and xenogeneic-free 3D protocol outlined within allows for the attainment of hPSC-derived microvascular-like ECs with high angiogenic potential. The absence of Matrigel enhances the translational capacity of the hPSC-MVECs arising from this protocol that could now be derived from patient-specific hPSC lines for evaluation in pre-clinical models of post-myocardial infarction cardiac regeneration or for in vivo models of cardiac remodelling and microvasculature disease.

The effect of antibody-mediated PCSK9 inhibitor on bare-metal stent implantation outcome

Abstract Introduction Percutaneous coronary intervention (PCI) is the gold standard for the management of acute coronary syndrome, and has been benefited from advances in pharmacotherapy and device innovation. Nevertheless, in-stent restenosis and stent thrombosis remain serious complications following PCI procedure with stent implantation. Proprotein convertase subtilisin kexin type 9 (PCSK9) is an enzyme known for its role in plasma cholesterol homeostasis. Efficiently reducing plasma cholesterol, PCSK9 inhibition recently became a pivotal treatment strategy for hypercholesterolemia. However, the effect of PCSK9 inhibition on stent implantation outcomes remains uninvestigated. Methods Carotid artery bare-metal stent (BMS) implantation was performed on C57Bl/6 mice, which then a parallel assessment of various inflammatory mediators was performed. To inhibit PCSK9, alirocumab was administered weekly to antiplatelet-treated mice started before stent implantation. Six weeks after the stent implantation procedure, animals were harvested and histomorphometric analysis was performed on stented arteries. In vitro cell migration and cellular senescence were also assessed on alirocumab-treated primary human aortic endothelial (EC) and vascular smooth muscle cells (VSMC). Results Compared to sham intervention, stent implantation was associated with increased expression of several inflammatory mediators, including PCSK9. Interestingly, the increase in PCSK9 level was confirmed in the stented vascular tissue, but not in plasma (Fig 1A). Following alirocumab treatment, PCSK9 inhibition results in an increased intimal hyperplasia in the stented vascular segment of alirocumab-treated animals compared to controls (Fig 1B). In vitro, alirocumab promoted migration and inhibited the onset of senescence in primary human VSMC, while blunted the migration and increased the senescence of EC. Conclusion The results of this study demonstrated that antibody-based PCSK9 inhibition worsen bare-metal stent implantation outcome in mice. This observed effect is mediated, in part, by the differential effect on VSMC and EC senescence, thus promoting in-stent intimal hyperplasia while blunting vascular healing. The herein-reported data warrant additional investigations concerning the use of PCSK9 inhibitors in patients undergoing PCI with stent implantation.

RNA surveillance by ADAR1 is required for vascular endothelial resilience

Abstract Introduction Long dsRNAs are recognised as danger-associated molecular patterns by the cytosolic innate immune sensors inducing thus innate immune responses. RNA editing is a post-transcriptional modification induced by the binding of Adenosine Deaminases Acting on RNA (ADARs) to double-stranded RNA structures (dsRNAs). We have recently reported that ADAR1 is the main RNA editor in endothelial cells (ECs) but the role of ADAR1 in vascular homeostatic processes remains yet elusive. Methods Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1 , Ifh1 or Tlr3 allele with either a Tie2-Cre or with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1 effects. Results EC-specific ADAR1 deletion resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-ADAR1 in life. Inducible EC-ADAR1 ablation in adult mice elicited premature sudden death due to a widespread lung vascular leakage and pleural effusion, reflecting a compromised EC barrier function. Mechanistically, silencing of endothelial ADAR1 disrupted long-to-short dsRNA metabolism witnessed by the striking accumulation of cytoplasmic long dsRNAs. Subsequently, the cytoplasmic accumulation of endogenous long dsRNAs induced aberrant levels of interferon-β and activation of innate immune system. Finally, activation of the cytosolic dsRNA sensors inflicted the dissociation of β-catenin from VE-cadherin in EC junctions, while, co-silencing of the dsRNA sensor, MDA5, was sufficient to restore the integrity of the ADAR1-deficient endothelium. While double EC-specific ADAR1 and TLR3 did not fully rescue the lethality of the EC-ADAR1 animals, in vivo experiments using double EC-specific ADAR1 and MDA5 KO mice fully rescued the phenotype. Validation experiments using murine vascular ECs isolated from the mouse strains of our studies confirmed our mechanistic and phenotypic findings. Conclusion Endogenous suppression of double-stranded RNA-induced MDA5 autoinflammatory signaling by the RNA editor ADAR1 is essential for the maintenance of vascular integrity in adult mice.

Inhibition of farnesylation of Ras-GTPases ameliorates inflammation-induced loss of endothelial barrier integrity and leukocyte transmigration

Abstract Background and Aims The family of small GTPases (Rho- and Ras-GTPases) is known to regulate endothelial cytoskeleton dynamics and hence barrier integrity. The role of Rho GTPases (RhoA and Rac1) in endothelial barrier integrity is well characterized; however, little is known about the role of Ras-GTPases. The activity of small GTPases is regulated by several mechanisms, including post-translational prenylation (farnesylation or geranylgeranylation). The Ras-GTPases are preferentially post-translationally farnesylated by farnesyl transferases (FTs). The aim of the present study was to investigate whether inhibition of farnesylation of Ras-GTPases protects loss of endothelial barrier integrity induced by inflammatory mediators. Methods and Results Treatment of human primary endothelial cells (ECs; HUVEC and lung microvascular ECs) with pharmacological inhibitor of farnesyl transferases (FTIs), LB42708 (LB) and tipifarnib, stabilized basal endothelial barrier function (albumin-flux) and abrogated thrombin-induced hyperpermeability in a concentration- and time-dependent manner reaching maximum at a concentration of 2 mmol/L after 12 h of incubation. These concentrations of the FTIs reduced farnesylation of all the Ras isoforms but not the geranylgeranylation of Rho GTPases. Likewise, LB abrogated the TNFa-induced leucocyte transendothelial migration (Trans-well assay). Interestingly, FTIs treatment caused an enhanced translocation of VE-cadherin to cell-cell junctions (confocal microscopy) without exerting any effect on the actin cytoskeleton or endothelial contractile machinery; mainly regulated by Rho GTPases. The Ca2+-switch experiments demonstrated an inhibition of VE-cadherin internalization. These data were reproduced by lentiviral-mediated stable over-expression of the prenylation-dead mutants of Ras isoforms but not RhoA or Rac1. Conclusion The novel data of the present study demonstrate that FTIs protect endothelial barrier function against inflammatory mediator-induced endothelial hyperpermeability. We propose that FTIs are novel drug candidates for the treatment of edematous conditions (such as lung edema) where endothelial barrier integrity is compromised.

Major Facilitator Superfamily Domain Containing 5 Inhibition Reduces Lipoprotein(a) Uptake and Calcification in Valvular Heart Disease.

High circulating levels of Lp(a) (lipoprotein[a]) increase the risk of atherosclerosis and calcific aortic valve disease, affecting millions of patients worldwide. Although atherosclerosis is commonly treated with low-density lipoprotein-targeting therapies, these do not reduce Lp(a) or risk of calcific aortic valve disease, which has no available drug therapies. Targeting Lp(a) production and catabolism may provide therapeutic benefit, but little is known about Lp(a) cellular uptake. Here, unbiased ligand-receptor capture mass spectrometry was used to identify MFSD5 (major facilitator superfamily domain containing 5) as a novel receptor/cofactor involved in Lp(a) uptake. Reducing MFSD5 expression by a computationally identified small molecule or small interfering RNA suppressed Lp(a) uptake and calcification in primary human valvular endothelial and interstitial cells. MFSD5 variants were associated with aortic stenosis (P=0.027 after multiple hypothesis testing) with evidence suggestive of an interaction with plasma Lp(a) levels. MFSD5 knockdown suppressing human valvular cell Lp(a) uptake and calcification, along with meta-analysis of MFSD5 variants associating with aortic stenosis, supports further preclinical assessment of MFSD5 in cardiovascular diseases, the leading cause of death worldwide.

Abstract 12494: Role of Ceramide in Estrogen-Induced Oxidative Stress in Endothelial Cells From Individuals Assigned Male at Birth

Compared to cis-males and females, risk for cardiovascular disease (CVD) is elevated in trans-females undergoing estrogen-containing gender affirming therapy. We have previously shown that continuous estrogen treatment of arterioles from healthy adults assigned male at birth promotes microvascular endothelial dysfunction, however the mechanism through which estrogen contributes to endothelial damage remains unknown. Estrogen increases expression of neutral sphingomyelinase (NSmase), an enzyme responsible for the production of ceramides. Ceramides are bioactive sphingolipids that can increase cellular levels of hydrogen peroxide (H 2 O 2 ). Together, we hypothesize that estrogen increases endothelial levels of oxidative stress through NSmase-mediated ceramide formation. Primary male human umbilical vein endothelial cells (HUVECs; pooled from n=3) were seeded in 8-well microscopy plates prior to treatments. Peroxy yellow 1 (PY1) was used to measure changes in endothelial H 2 O 2 . Wells were imaged at 20x and analyzed using ImageJ. Difference between %change in fluorescence ±standard error (SE) is reported, and significance is determined using One-Way ANOVA. Treatment with 17β-estradiol (E2; 100nM, 48hrs) increased endothelial H 2 O 2 production (control, n=6 vs E2, n=7; 13.9±6.3, p=0.038), an effect diminished in the presence of the NSmase inhibitor GW4869 (GW4869, 4μM, 48hrs; control, n=6 vs E2+GW4869, n=7; 1.7±6.3, p=0.78). Signal specificity for H 2 O 2 was confirmed with PEG-catalase treatment (500U/ml, 48hrs; control, n=6 vs E2+catalase, n=3; 2.3±7.9, p=0.77). Together, these results suggest that estrogen increases oxidative stress in endothelial cells isolated from individuals assigned male at birth which can be prevented through inhibition of the ceramide-forming enzyme NSmase. This work offers mechanistic insight into how estrogen containing gender affirming therapy may potentially contribute to endothelial damage and increase CVD risk in trans-females.

Targeting BRD4 to Counteract Cytokine-Induced Expression of PD-L1 and PD-L2 on Vascular, Osteoblastic and Stromal Niche Cells

The stem cell niche plays a crucial role in promoting the expansion of leukemic stem cells (LSC) and in mediating resistance of LSC against various anti-neoplastic drugs in different types of leukemias. Recent data suggest that targeting niche cells may be an effective approach to overcome niche-mediated drug resistance of LSC. However, niche cells themselves are considered to be often resistant against therapeutic intervention. This may especially be relevant in those myeloid neoplasms where niche cell expansion is a pathognomonic feature and the resulting neo-angiogenesis, bone marrow fibrosis and/or sclerosis are important prognostic variables. We and others have recently shown that several of the anti-neoplastic drugs used to treat myeloid leukemias can also counteract niche cell expansion. So far, however, little is known about mechanisms underlying resistance of niche cells against anti-neoplastic drugs. Recent studies have shown that certain immune checkpoint antigens are overexpressed in cancer cells and/or their microenvironment and that these checkpoint antigens contribute to drug resistance. Other studies have shown that checkpoint molecules, including PD-L1 and CD47, are displayed by LSC in acute myeloid leukemia, chronic myeloid leukemia and other myeloid neoplasms. We asked whether niche-related cells also display these checkpoint molecules. Furthermore, we screened for cytokines that promote expression of checkpoint antigens on niche cells and asked for drugs that counteract growth and cytokine-induced checkpoint expression in these cells. The following niche cells were examined: primary human umbilical vein endothelial cells (HUVEC), the human endothelial cell line HMEC-1, the human osteoblast-like cell line CAL-72, primary osteoblasts, and the human stromal cell line HS5. Expression of the checkpoint antigens PD-L1, PD-L2, PD1, CD28, CD47, CD80, CD83, CD86 and TIM-3 was determined by flow cytometry. All niche cells were found to constitutively express PD-L1, PD-L2, and CD47. The other checkpoint antigens were expressed at low or undetectable levels in these cells. Expression of PD-L1, PD-L2 and CD47 in niche cells was confirmed by quantitative PCR and by immunocytochemistry. Of all cytokines tested (n=12), interferon-alpha (IFN-A; effective range 50-500 ng/ml) and interferon-gamma (IFN-G; 10-500 ng/ml) were found to promote surface expression of PD-L1 and PD-L2 in HMEC-1, CAL72, and HS5 cells in our immunocytochemical and flow cytometry experiments. Moreover, we found that tumor necrosis factor-alpha (TNF-A; 100 ng/ml) enhances IFN-G-induced expression of PD-L1 and PD-L2 in HMEC-1, CAL-72 and HS5 cells. The BRD4-targeting drug JQ1 and the BRD4 degraders dBET1 and dBET6 suppressed cytokine-induced upregulation of surface expression of PD-L1 and PD-L2 in HMEC-1, HUVEC, CAL-72 and HS5 cells, suggesting a role for BRD4 and MYC in checkpoint regulation. In all cell lines examined, dBET6 was found to be a more effective drug compared to JQ1 and dBET1 (IFN-G-induced expression of PD-L1 in: HMEC-1: IC 50 for JQ1 0.5 µM vs dBET1 1 µM vs dBET6 0.05 µM; CAL-72: IC 50 for JQ1 >0.5 µM vs dBET1 1 µM vs dBET6 <0.05 µM). Together, we show that endothelial cells, osteoblasts and stromal cells display PD-L1, PD-L2 and CD47 in a constitutive manner. Our data also show that the pro-inflammatory cytokines IFN-G and IFN-A augment expression of PD-L1 and PD-L2 in these cells, and that TNF-A can further enhance IFN-A/IFN-G-induced expression of PD-L1 and PD-L2 in niche cells. In addition, we show that the BRD4-targeting drugs JQ1, dBET1 and dBET6 suppress cytokine-induced PD-L1 and PD-L2 expression on niche cells. Whether checkpoint expression in niche cells is associated with drug resistance in myeloid neoplasms and whether drug-induced suppression of checkpoint expression in niche cells can overcome resistance remains unknown.

Fibrinogen in mice cerebral microvessels induces blood–brain barrier dysregulation with aging via a dynamin-related protein 1–dependent pathway

We previously reported evidence that oxidative stress during aging leads to adverse protein profile changes of brain cortical microvessels (MVs: end arterioles, capillaries, and venules) that affect mRNA/protein stability, basement membrane integrity, and ATP synthesis capacity in mice. As an extension of our previous study, we also found that proteins which comprise the blood–brain barrier (BBB) and regulate mitochondrial quality control were also significantly decreased in the mice’s cortical MVs with aging. Interestingly, the neuroinflammatory protein fibrinogen (Fgn) was increased in mice brain MVs, which corresponds with clinical reports indicating that the plasma Fgn concentration increased progressively with aging. In this study, protein–protein interaction network analysis indicated that high expression of Fgn is linked with downregulated expression of both BBB- and mitochondrial fission/fusion–related proteins in mice cortical MVs with aging. To investigate the mechanism of Fgn action, we observed that 2 mg/mL or higher concentration of human plasma Fgn changed cell morphology, induced cytotoxicity, and increased BBB permeability in primary human brain microvascular endothelial cells (HBMECs). The BBB tight junction proteins were significantly decreased with increasing concentration of human plasma Fgn in primary HBMECs. Similarly, the expression of phosphorylated dynamin-related protein 1 (pDRP1) and other mitochondrial fission/fusion–related proteins were also significantly reduced in Fgn-treated HBMECs. Interestingly, DRP1 knockdown by shRNA(h) resulted in the reduction of both BBB- and mitochondrial fission/fusion–related proteins in HBMECs. Our results suggest that elevated Fgn downregulates DRP1, leading to mitochondrial-dependent endothelial and BBB dysfunction in the brain microvasculature.