Aim/hypothesis
Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation ...status in type 2 diabetes, which is characterised by chronic inflammation.
Methods
We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68
+
CCR2
+
) and M2 (CX3CR1
+
CD206
+
/CD163
+
) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals.
Results
We found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA
1c
and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals.
Conclusions/interpretation
We show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.
Abstract
Background
Coronary artery disease (CAD) and cardiovascular diseases (CVD) in general still represent the leading cause of death worldwide. As a multifactorial disease entity, genetic ...predisposition and lifestyle strongly impact disease development and progression. Here, induced pluripotent stem cells (iPSC) are an ideal tool to differentiate (epi-)genetic from lifestyle disease risk modulation and identify the underlying molecular mechanisms.
Aim
This study aims to determine differences in iPSC-derived endothelial cell (iPSC-EC) characteristics and functionality between healthy donors and high-risk CAD patients.
Methods
IPSC from participants with similar lifestyle but different disease status (healthy >65 y/o, n=5, acute coronary syndrome (ACS) <65y/o, n=6) were differentiated into iPSC-EC. Cells were exposed to inflammatory stimulation with tumour necrosis factor alpha (TNF-a) followed by bulk RNA-seq. Monocyte (THP-1) recruitment was assessed under protective and atherogenic flow conditions. Mitotic age was calculated based on DNA-methylation via Hannum estimate (1) for each donor at the iPSC and iPSC-EC (passage 4) stage.
Results
ACS-iPSC-EC had significantly higher upregulation of E-selectin upon TNF-a stimulation on mRNA level (ACS: 532.1-fold±48.7 vs healthy: 322.3-fold±55.7 with p=0.02). Similarly, ICAM1 protein upregulation was higher in ACS-iPSC-EC compared to healthy iPSC-EC as determined by flow cytometry (ACS: 1.4-fold±0.01 vs healthy: 1.1-fold±0.0007; p<0.001). ACS-iPSC-EC recruited significantly more THP-1 per view field under protective (high shear stress, laminar) flow conditions (ACS: 78.4±20.7 vs healthy: 33.25±12.3 with p=0.04) compared to healthy iPSC-EC. While both groups recruited significantly more THP-1 under atherogenic than protective conditions (ACS: 302±38.9 with p=0.0055 vs healthy: 340±70.4 with p=0.0005), there was no difference between the donor groups (p=0.43). Analysis of bulk RNA-seq data showed significant differences in TNF-a-induced transcriptome changes. Gene set enrichment analysis identified differences in signalling pathways related to leukocyte and lymphocyte-mediated immunity, adaptive immune response, cytokine activity, and many more as shown in Figure 1 (p.adj ≤1e-10). IPSC of both groups had a negative calculated mitotic age (in years) which significantly increased after endothelial differentiation and four population doublings (ACS-iPSC: −14.1±0.7 vs ACS-EC: −0.1±1.1, p<0.0001 and healthy-iPSC: −13.7±0.8 vs healthy-EC: 2.5±1.5, p=0.0001).
Conclusion
The higher rate of basal THP-1 recruitment and upregulation of adhesion molecules on mRNA and protein level and the distinct transcriptome changes suggest intrinsic differences between patient and healthy iPSC-EC both in the basal state and in inflammatory response. Furthermore, this demonstrates iPSC as a viable in vitro model also for polygenetic diseases such as CAD.
Funding Acknowledgement
Type of funding sources: Foundation. Main funding source(s): German Cardiac Society, Research Scholarship
Background and purpose:
Mature endothelial cells and their progenitors are dysfunctional in diabetes, resulting in deficient neovascularisation following arterial occlusion. This study aimed to ...evaluate the therapeutic activity of a nitric oxide (NO) releasing statin in the setting of experimental diabetes and peripheral ischaemia.
Experimental approach:
The effects of NCX 6550, an NO‐releasing pravastatin derivative, on angiogenesis in ischaemic limbs was studied in normoglycaemic mice or mice made diabetic by treatment with streptozotocin (STZ). Control mice received an equimolar dosage of the parent statin compound, pravastatin. The therapeutic action of NCX 6550 was also tested in mice lacking the gene for endothelial nitric oxide synthase (eNOS).
Key Results:
In normoglycaemic or STZ‐diabetic CD1 mice, only NCX 6550 stimulated skeletal muscle revascularisation. In addition, NCX 6550 induced greater improvement in limb reperfusion and salvage, than pravastatin. The number of circulating endothelial progenitor cells was decreased in STZ‐diabetic mice, this defect being prevented by NCX 6550 and, to a lesser extent by pravastatin. In vitro, high glucose concentrations reduced the migratory capacity of endothelial progenitor EPCs, which was partly reversed by preincubation with pravastatin and completely reversed by NCX 6550. The postischaemic recovery of eNOS knockout mice was severely impaired as a consequence of depressed angiogenesis and this recovery was improved by treatment with NCX 6550, but not with pravastatin.
Conclusions and implications:
These findings indicate that incorporation of a bioactive NO moiety improves the therapeutic profile of statins for the treatment of peripheral vascular disease.
British Journal of Pharmacology (2007) 150, 873–882. doi:10.1038/sj.bjp.0707142
Over the past two decades, extensive research has focused on arterial remodelling in both physiological and pathological ageing. The concept now describes the growth as well as the rearrangement of ...cellular components and extracellular matrix, resulting in either reduction or increase in vessel lumen. In diabetes, remodelling extends to capillaries, microvascular beds, and arteries of different calibre. This process is paralleled by accelerated atherosclerosis and accounts for an increased incidence of ischaemic complications. The incapacity of pre-existing and de novo formed collaterals to bypass atherosclerotic occlusions, combined with a decline in tissue capillary density, is responsible for the delayed recovery from ischaemia and ultimately leads to organ failure. The mechanisms of vascular remodelling are incompletely understood, but metabolic and mechanical factors seem to play an important role. Hyperglycaemia represents the main factor responsible for the fast progression of atherosclerosis as well as microangiopathy. However, intensive blood glucose control alone is insufficient to reduce the risk of macrovascular complications. Pharmacological control of oxidative stress and stimulation of nitric oxide release have proved to exert beneficial effects on vascular remodelling in experimental diabetic models. New approaches of regenerative medicine using vascular progenitor cells for the treatment of ischaemic disease have been shown to be safe and are now being tested for efficacy in pre-clinical and clinical trials.
Abstract
Background
Hemostasis is dysregulated in patients with moderate-to-severe coronavirus disease 2019 (COVID-19). However, patients with respiratory diseases other than COVID-19 can also show ...disturbed coagulation reactions during the acute inflammatory process. Parameters of coagulation and platelet function are here compared between patients with upper respiratory infections with and without COVID-19 and are related to the clinical outcome.
Methods
Hospitalized patients with acute respiratory symptoms and with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-positive (COVpos) and SARS-CoV2-negative (COVneg) status were included. We assessed several parameters of coagulation and fibrinolysis as well as adenosine diphosphate (ADP)-, thrombin receptor activator peptide 6 (TRAP)-, and arachidonic acid (AA)-induced platelet reactivity by impedance aggregometry, as well as leukocyte subtype spectrum and platelet-leukocyte aggregates by flow cytometry and inflammatory cytokines by cytometric bead array. The SOFA score was assessed as marker of the clinical outcome.
Results
87 patients were included in the study of which 50 were COVpos and 37 were COVneg. Von Willebrand factor was significantly higher in COVID positive patients compared to the control group (4456,4 mU/ml 2701,4; 9067,0 vs. 2528,0 mU/ml 1301,2; 3693,8, p<0,001). COVID-positive patients exhibited also more tissue plasminogen activator in the circulating blood than COVID-negative patients with an respiratory infection (11,4 mU/ml 7,2; 24,6 vs. 7,3 mU/ml 4,9; 10,5, p=0,001). ADP, TRAP-, and AA-induced platelet reactivity was significantly higher in COVpos than in COVneg patients. The SOFA score was higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. The SOFA score correlated significantly with parameters of coagulation and platelet function. A larger percentage of classical and intermediate monocytes, and of CD4pos T cells (TH) aggregated with platelets in COVpos than in COVneg patients. Interleukin (IL)-1 receptor antagonist (RA) and IL-6 levels were higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. IL-1RA and IL-6 levels correlated with the SOFA score in COVpos but not in COVneg patients, indicating a COVID-19-specific mechanism.
Conclusion
In moderate-to-severe COVID-19, but not in other respiratory diseases, disease severity was associated with parameters of coagulation and platelet function. Dysregulated coagulation and platelet hyperreactivity were associated to a worse clinical outcome in patients with COVID-19, pointing to the importance of antithrombotic therapy for reducing disease severity.
Funding Acknowledgement
Type of funding sources: None.
Abstract
Background
Galectin-3 is a β-galactoside-binding lectin that has been described to be overexpressed in inflammation, atherosclerosis, and in myocardial fibrosis. In COVID-19, galectin-3 has ...been proposed as an important regulator of the inflammatory response and fibrosis processes. The role of galectin-3 as a platelet activator and thrombosis enhancer has been also recently described. However, the role of galectin-3 in the thrombotic risk in COVID-19 hasn't been studied extensively.
Methods
Patients with moderate to severe COVID-19 were included in the study. Hospitalized patients with acute respiratory diseases without COVID-19 were examined as controls. We compared the levels of galectin-3, soluble ST2, tissue factor and tissue factor activity (TFa) as well as several other markers of increased thrombogenicity in both groups. The correlations between galectin-3 and coagulation as well as inflammation markers were assessed. The SOFA score was used as a marker for the clinical outcome.
Results
93 patients were included into the study of which 56 were SARS-CoV-2 positive (COV+) and 37 were SARS-CoV-2 negative controls (COV−). Galectin-3 levels were higher in the COV+ group (median 7.10 ng/ml IQR 4.61–9.81 vs. 5.47 ng/ml 3.63–6.66 p=0.016) as well as the TFa (median 334.48 pM 115.19–632.58 vs. 134.02 pM 86.92–206.66) and the ST2 levels (median 5.49 ng/ml 2.40–9.28 vs. 2.19 ng/ml 0.66–3.91 p<0.001). We also observed a positive correlation between galectin-3 and IL-6 (r=0.559, p<0.001), ST2 (r=0.332, p=0.005), SOFA score (r=0.441, p=0.003), von Willebrand factor (r=0.401, p<0.001), plasminogen (r=0.361, p=0.001), antithrombin (r=0.453, p<0.001), and D-dimer (r=0.377, p=0.001).
Conclusion
In patients with acute respiratory diseases, especially with COVID-19, galectin-3 is a marker for increased hypercoagulability and worse clinical outcome. Galactin-3 might be a useful therapeutic target for patients with COVID-19.
Funding Acknowledgement
Type of funding sources: None.
Abstract
Background
Inflammation is a protective response triggered not only upon pathogen insults but also- in sterile injury as happening during hypoxic episodes in acute myocardial infarction ...(AMI). Monocytes are among the first responder immune cells playing an important role in inflammation induction but also in its timely resolution. Understanding the mechanisms governing this two-sided modulation can help to uncover novel therapeutic approaches.
Inflammasomes are crucial gatekeepers of the immune response, but their maladaptive activation associates with inflammatory pathologies. Besides canonical activation, monocytes can trigger non-transcriptional or rapid inflammasome activation. In this project, we defined rapid inflammasome activation (by simultaneous TLR priming and ATP stimulation) in the context of AMI and examined its regulation.
Methods
Human blood was collected in EDTA tubes according to ethical standards and upon consent of the included patients. The three main monocyte subpopulations, defined as classical, intermediate and non-classical were sorted on a FACS Aria II based on the expression of the surface markers CD14 and CD16. We measured caspase-1 (CASP1) activity in monocyte subpopulations with FLICA Assay and quantified interleukin-1β and interleukin-18 release via ELISA. RNA and protein levels of inflammasome associated genes were determined by real-time PCR and Western blotting. We also measured hsTnT in plasma of AMI patients with ELISA.
Results
Circulating monocytes from AMI patients, especially the classical subpopulation, displayed strong attenuation of CASP1 activity but not total since inflammasome inhibition (MCC950) reduced CASP1 activation further. Rapid activation of NLRP3 induced acute IL18 release, mostly in classical monocytes of healthy subjects. IL18 release was also reduced in monocytes obtained from AMI patients compared to healthy. Blood circulating monocytes seemed to be primed by DAMPs released from the ischemic lesion. This induced transcriptional reprogramming inducing upregulation of TNFa induced protein 3 (TNFAIP3) and IRAKM, two early response genes, although at different time scales. TNFAIP3 protein levels positively associated with cardiac injury measured via hsTnT levels in plasma. We also observed upregulation of TNFAIP3-AS in AMI patients, which is the lncRNA antisense in the TNFAIP3 loci.
Conclusion and Future Approaches
Circulating monocytes in AMI present a phenotype with attenuated inflammasome activation and IL18 release. TNFAIP3 expression correlates with cardiac injury (hsTnT) and acts as a potential modulator of inflammasome activity in a protective mechanism against hyper-activation of systemic inflammation . In the future we aim to define the mechanisms of TNFAIP3 and its splicing isoforms in the attenuation of inflammasome activation as well as the function of TNFAIP3 antisense in the regulation of this response.
Abstract
Background
Increased systemic inflammation and metabolic dysfunction are observed in heart failure with reduced ejection fraction (HFrEF). On the other hand, cardiorespiratory exercise ...testing (CPET) exerts a physical challenge and initiates the activation of the immune system, including acute release of natural killer (NK) cells into the circulation, and several metabolic pathways.
Aim
To characterize the inflammatory and metabolic alterations of HFrEF patients in response to an acute exercise challenge, and after 2 hours of recovery.
Methods
Participants with HFrEF (n=16), age and sex matched controls (CON, n=13) were investigated at baseline, immediately after and 2 hours after CPET. Clinical and physiological parameters, leukocyte profile, plasma cytokines and metabolites were assessed along with inflammatory and metabolic parameters at all three time points. NK cell counts and morphological/activation parameters in different contexts were examined. Further, the time-dependent coordination of NK cell numbers post-exercise with tryptophan metabolism and plasma triglycerides were assessed. NK cells were isolated from blood of healthy donors for ex vivo proof-of-principle experiments, including phenotype polarization and NK cell specific tryptophan metabolism.
Results
Cardiovascular risk profiles as well as leukocyte, cytokine and metabolic parameters at baseline were similar in CON and HFrEF. Immediately after CPET, lactate, and NK T cell blood counts were significantly increased in both groups. In HFrEF but not CON, platelet aggregates with NK cells, CD8+ cytotoxic T cells and “classical” CD14++CD16-monocytes, phosphatidylcholines and triglycerides were increased. After 2h of recovery, almost all altered parameters returned to baseline in CON. In contrast, blood counts and morphological markers of inflammatory effector cell types, including CD8+ T cells and neutrophils remained elevated in HFrEF. NK cells remained elevated after the recovery period and correlated with levels of various triglyceride species in the HFrEF patients. Tryptophan levels in plasma were reduced by acute exercise and the kynurenine to tryptophan ratio was increased and correlated with increase in NK and NK-T cell counts, as well as IL-12 plasma levels. Treatment with IL-12 led to increased synthesis of kynurenine from tryptophan, expression of indoleamine 2,3-dioxygenase and abundance of regulatory CD56bri NK cell phenotypes ex vivo. Secretome of untreated NK cells impaired cellular respiration, increased glycolysis/oxidation ratio in skeletal muscle cells, and increased the release of triglycerides from hepatocarcinoma cells.
Conclusion
CPET induced a complex acute immunometabolic response, whose restitution to baseline levels differed between HFrEF and healthy controls. Exercise-induced changes in NK cell metabolism and phenotype shift might modulate cellular respiration in myocytes and release of triglycerides by hepatocytes in HFrEF and in CON.
Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DZHK (German Centre for Cardiovascular Research)
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