Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary... Show moreChronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-kappa B pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1 beta, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-kappa B signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-kappa B inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-kappa B inhibitor. To determine if NF-kappa B was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-kappa B-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-kappa B-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-kappa B signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-kappa B signaling and potentially contributes to the pathophysiology and progression of CTEPH. Show less
Hoogland, I.C.M.; Westhoff, D.; Engelen-Lee, J.Y.; Seron, M.V.; Houben-Weerts, J.H.M.P.; Westerloo, D.J. van; ... ; Beek, D. van de 2021
Systemic infection is an important risk factor for the development cognitive impairment and neurodegeneration in older people. Animal experiments show that systemic challenges with live bacteria... Show moreSystemic infection is an important risk factor for the development cognitive impairment and neurodegeneration in older people. Animal experiments show that systemic challenges with live bacteria cause a neuro-inflammatory response, but the effect of age on this response in these models is unknown. Young (2 months) and middle-aged mice (13-14 months) were intraperitoneally challenged with live Escherichia coli (E. coli) or saline. The mice were sacrificed at 2, 3 and 7 days after inoculation; for all time points, the mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h after inoculation. Microglial response was monitored by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry, and inflammatory response by mRNA expression of pro- and anti-inflammatory mediators. We observed an increased microglial cell number and moderate morphologically activated microglial cells in middle-aged mice, as compared to young mice, after intraperitoneal challenge with live E. coli. Flow cytometry of microglial cells showed higher CD45 and CD11b expressions in middle-aged infected mice compared to young infected mice. The brain expression levels of pro-inflammatory genes were higher in middle-aged than in young infected mice, while middle-aged infected mice had similar expression levels of these genes in the systemic compartment. We conclude that systemic challenge with live bacteria causes an age-dependent neuro-inflammatory and microglial response. Our data show signs of an age-dependent disconnection of the inflammatory transcriptional signature between the brain and the systemic compartment. Show less
Putten, C. van der; Remmerswaal, E.B.M.; Terpstra, M.L.; Bom, N.D. van der; Kers, J.; Berge, I.J.M. ten; ... ; Aalderen, M.C. van 2021
Background: At border sites, and in internal organs, tissue resident memory T cells (T-RM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However,... Show moreBackground: At border sites, and in internal organs, tissue resident memory T cells (T-RM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However, information on the presence and function of these cells in the human kidney is scant. In order to better understand the T cell-mediated immunological defense in this organ, we aimed to determine phenotypic and functional aspects of CD8 and CD4 T cells present in healthy and allograft kidney tissue. Methods: Using multichannel flow cytometry, we assessed the phenotype and function of T cells in healthy renal tissue samples (n = 5) and kidney allograft tissue (n = 7) and compared these aspects to T cells in peripheral blood from healthy controls (n = 13). Results: Kidney tissue samples contained substantial amounts of CD8 and CD4 T cells. In contrast to the circulating cells, kidney T cells frequently expressed CD69 and CD103, and were more often actively cycling. Furthermore, nearly all kidney T cells expressed CXCR3, and often expressed CXCR6 compared to T cells in the circulation. Markedly, kidney T cells produced greater quantities of IFN gamma than circulating cells and were frequently polyfunctional. Conclusion: Functional T cells with the characteristic traits of T-RM reside in human kidney tissues. These cells are more often actively cycling and frequently express CXCR3 and CXCR6. Show less
Gracht, E.T.I. van der; Behr, F.M.; Arens, R. 2021
Tissue-resident memory T (T-RM) cells mediate potent local innate and adaptive immune responses and provide long-lasting protective immunity. T-RM cells localize to many different tissues,... Show moreTissue-resident memory T (T-RM) cells mediate potent local innate and adaptive immune responses and provide long-lasting protective immunity. T-RM cells localize to many different tissues, including barrier tissues, and play a crucial role in protection against infectious and malignant disease. The formation and maintenance of T-RM cells are influenced by numerous factors, including inflammation, antigen triggering, and tissue-specific cues. Emerging evidence suggests that these signals also contribute to heterogeneity within the T-RM cell compartment. Here, we review the phenotypic and functional heterogeneity of CD8(+) T-RM cells at different tissue sites and the molecular determinants defining CD8(+) T-RM cell subsets. We further discuss the possibilities of targeting the unique cell surface molecules, cytokine and chemokine receptors, transcription factors, and metabolic features of T-RM cells for therapeutic purposes. Their crucial role in immune protection and their location at the frontlines of the immune defense make T-RM cells attractive therapeutic targets. A better understanding of the possibilities to selectively modulate T-RM cell populations may thus improve vaccination and immunotherapeutic strategies employing these potent immune cells. Show less
Recent translational studies highlighted the inhibition of transforming growth factor (TGF)-beta signaling as a promising target to treat pulmonary arterial hypertension (PAH). However, it remains... Show moreRecent translational studies highlighted the inhibition of transforming growth factor (TGF)-beta signaling as a promising target to treat pulmonary arterial hypertension (PAH). However, it remains unclear whether alterations in TGF-beta signaling are consistent between PAH patients and animal models. Therefore, we compared TGF-beta signaling in the lungs of PAH patients and rats with experimental PAH induced by monocrotaline (MCT) or SU5416+hypoxia (SuHx). In hereditary PAH (hPAH) patients, there was a moderate increase in both TGF beta R2 and pSMAD2/3 protein levels, while these were unaltered in idiopathic PAH (iPAH) patients. Protein levels of TGF beta R2 and pSMAD2/3 were locally increased in the pulmonary vasculature of PAH rats under both experimental conditions. Conversely, the protein levels of TGF beta R2 and pSMAD2/3 were reduced in SuHx while slightly increased in MCT. mRNA levels of plasminogen activator inhibitor (PAI)-1 were increased only in MCT animals and such an increase was not observed in SuHx rats or in iPAH and hPAH patients. In conclusion, our data demonstrate considerable discrepancies in TGF beta-SMAD signaling between iPAH and hPAH patients, as well as between patients and rats with experimental PAH. Show less
As heart failure (HF) is a devastating health problem worldwide, a better understanding and the development of more effective therapeutic approaches are required. HF is characterized by sympathetic... Show moreAs heart failure (HF) is a devastating health problem worldwide, a better understanding and the development of more effective therapeutic approaches are required. HF is characterized by sympathetic system activation which stimulates alpha- and beta-adrenoceptors (ARs). The exposure of the cardiovascular system to the increased locally released and circulating levels of catecholamines leads to a well-described downregulation and desensitization of beta-ARs. However, information on the role of alpha-AR is limited. We have performed a systematic literature review examining the role of both cardiac and vascular alpha(1)-ARs in HF using 5 databases for our search. All three alpha(1)-AR subtypes (alpha(1A), alpha(1B) and alpha(1D)) are expressed in human and animal hearts and blood vessels in a tissue-dependent manner. We summarize the changes observed in HF regarding the density, signaling and responses of alpha(1)-ARs. Conflicting findings arise from different studies concerning the influence that HF has on alpha(1)-AR expression and function; in contrast to beta-ARs there is no consistent evidence for down-regulation or desensitization of cardiac or vascular alpha(1)-ARs. Whether alpha(1)-ARs are a therapeutic target in HF remains a matter of debate. Show less
Muñoz-Sánchez, S.; Vaart, M. van der; Meijer, A.H. 2020
Hematopoietic stem cells (HSCs) develop at several anatomical locations and are thought to undergo different niche regulatory cues originating from highly conserved cell signaling pathways, such as... Show moreHematopoietic stem cells (HSCs) develop at several anatomical locations and are thought to undergo different niche regulatory cues originating from highly conserved cell signaling pathways, such as Wnt, Notch, TGF-beta family, and Hedgehog signaling. Most insight into these pathways has been obtained by reporter models and loss- or gain of function experiments, yet results differ in many cases according to the approach. In this review, we discuss existing murine reporter models regarding these pathways, considering the genetic constructs and reporter proteins in the context of HSC studies; yet these models are relevant for all other stem cell systems. Lastly, we describe a multi-reporter model to properly study and understand the cross-pathway interaction and how reporter models are highly valuable tools to understand complex signaling dynamics in stem cells. Show less
The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements... Show moreThe ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined results strongly suggest that CPXV012 peptide inhibits virus infections by direct interactions with phosphatidylserine in the viral envelope. These results reiterate the potential of cationic peptides as broadly-acting virus inhibitors. Show less
Background:Mutations in bone morphogenetic protein receptor type II (BMPR2) are leading to the development of hereditary pulmonary arterial hypertension (PAH). In non-hereditary forms of PAH,... Show moreBackground:Mutations in bone morphogenetic protein receptor type II (BMPR2) are leading to the development of hereditary pulmonary arterial hypertension (PAH). In non-hereditary forms of PAH, perturbations in the transforming growth factor-beta (TGF-beta)/BMP-axis are believed to cause deficient BMPR2 signaling by changes in receptor expression, the activity of the receptor and/or downstream signaling. To date, BMPR2 expression and its activity in the lungs of patients with non-hereditary PAH is poorly characterized. In recent decades, different animal models have been used to understand the role of BMPR2 signaling in PAH pathophysiology. Specifically, the monocrotaline (MCT) and Sugen-Hypoxia (SuHx) models are extensively used in interventional studies to examine if restoring BMPR2 signaling results in PAH disease reversal. While PAH is assumed to develop in patients over months or years, pulmonary hypertension in experimental animal models develops in days or weeks. It is therefore likely that modifications in BMP and TGF-beta signaling in these models do not fully recapitulate those in patients. In order to determine the translational potential of the MCT and SuHx models, we analyzed the BMPR2 expression and activity in the lungs of rats with experimentally induced PAH and compared this to the BMPR2 expression and activity in the lungs of PAH patients.Methods:the BMPR2 expression was analyzed by Western blot analysis and immunofluorescence (IF) microscopy to determine the quantity and localization of the receptor in the lung tissue from normal control subjects and patients with hereditary or idiopathic PAH, as well as in the lungs of control rats and rats with MCT or SuHx-induced PAH. The activation of the BMP pathway was analyzed by determining the level and localization of phosphorylated Smad1/5/8 (pSmad 1/5/8), a downstream mediator of canonical BMPR2 signaling.Results:While BMPR2 and pSmad 1/5/8 expression levels were unaltered in whole lung lysates/homogenates from patients with hereditary and idiopathic PAH, IF analysis showed that BMPR2 and pSmad 1/5/8 levels were markedly decreased in the pulmonary vessels of both PAH patient groups. Whole lung BMPR2 expression was variable in the two PAH rat models, while in both experimental models the expression of BMPR2 in the lung vasculature was increased. However, in the human PAH lungs, the expression of pSmad 1/5/8 was downregulated in the lung vasculature of both experimental models.Conclusion:BMPR2 receptor expression and downstream signaling is reduced in the lung vasculature of patients with idiopathic and hereditary PAH, which cannot be appreciated when using human whole lung lysates. Despite increased BMPR2 expression in the lung vasculature, the MCT and SuHx rat models did develop PAH and impaired downstream BMPR2-Smad signaling similar to our findings in the human lung. Show less
Following fifteen years of research, neutrophil extracellular traps (NETs) are widely reported in a large range of inflammatory infectious and non-infectious diseases. Cumulating evidences from in... Show moreFollowing fifteen years of research, neutrophil extracellular traps (NETs) are widely reported in a large range of inflammatory infectious and non-infectious diseases. Cumulating evidences from in vitro, in vivo and clinical diagnostics suggest that NETs may play a crucial role in inflammation and autoimmunity in a variety of autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Most likely, NETs contribute to breaking self-tolerance in autoimmune diseases in several ways. During this review, we discuss the current knowledge on how NETs could drive autoimmune responses. NETs can break self-tolerance by being a source of autoantigens for autoantibodies found in autoimmune diseases, such as anti-citrullinated protein antibodies (ACPAs) in RA, anti-dsDNA in SLE and anti-myeloperoxidase and anti-protein 3 in AAV. Moreover, NET components could accelerate the inflammatory response by mediating complement activation, acting as danger-associated molecular patterns (DAMPs) and inflammasome activators, for example. NETs also can activate other immune cells, such as B cells, antigen-presenting cells and T cells. Additionally, impaired clearance of NETs in autoimmune diseases prolongs the presence of active NETs and their components and, in this way, accelerate immune responses. NETs have not only been implicated as drivers of inflammation, but also are linked to resolution of inflammation. Therefore, NETs may be central regulators of inflammation and autoimmunity, serve as biomarkers, as well as promising targets for future therapeutics of inflammatory autoimmune diseases. Show less
Brescia, M.; Janssen, J.M.; Liu, J.; Goncalves, M.A.F.V. 2020
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the similar to 2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs)... Show moreDuchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the similar to 2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% +/- 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of beta-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD-correcting agents. Show less
Gene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and... Show moreGene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and differentiation capabilities consent studying cellular differentiation processes, screening small-molecule drugs, modeling human disorders, and testing regenerative medicines. To integrate gene editing and stem cell technologies, there is a critical need for achieving efficient delivery of the necessary molecular tools in the form of programmable DNA-targeting enzymes and/or exogenous nucleic acid templates. Moreover, the impact that the delivery agents themselves have on the performance and precision of gene editing procedures is yet another critical parameter to consider. Viral vectors consisting of recombinant replication-defective viruses are under intense investigation for bringing about efficient gene-editing tool delivery and precise gene-editing in human cells. In this review, we focus on the growing role that adenoviral vectors are playing in the targeted genetic manipulation of human stem cells, progenitor cells, and their differentiated progenies in the context of in vitro and ex vivo protocols. As preamble, we provide an overview on the main gene editing principles and adenoviral vector platforms and end by discussing the possibilities ahead resulting from leveraging adenoviral vector, gene editing, and stem cell technologies. Show less
We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such... Show moreWe recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia. Show less
Chen, L.; Boleslaw, O.M.; Kruithof-de Julio, M.; Snaar-Jagalska, B.E. 2020
Mast cells have been associated with arteriogenesis and collateral formation. In advancedhuman atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels,and mast... Show moreMast cells have been associated with arteriogenesis and collateral formation. In advancedhuman atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels,and mast cells have also been associated with tumor vascularization. Based on these associations,we hypothesize that mast cells promote angiogenesis during ischemia. In human ischemic muscletissue from patients with end-stage peripheral artery disease, we observed activated mast cells,predominantly located around capillaries. Also, in mouse ischemic muscles, mast cells were detectedduring the revascularization process and interestingly, mast cell activation status was enhanced up to10 days after ischemia induction. To determine whether mast cells contribute to both arteriogenesisand angiogenesis, mast cells were locally activated immediately upon hind limb ischemia in C57Bl/6mice. At day 9, we observed a 3-fold increase in activated mast cell numbers in the inguinal lymphnodes. This was accompanied by an increase in the amount of Ly6Chigh inflammatory monocytes.Interestingly, local mast cell activation increased blood flow through the hind limb (46% at day 9)compared to that in non-activated control mice. Histological analysis of the muscle tissue revealedthat mast cell activation did not aect the number of collaterals, but increased the collateral diameter,as well as the number of CD31+ capillaries. Together, these data illustrate that locally activated mastcell contribute to arteriogenesis and angiogenesis. Show less
