Kalle Sipilä

Molecular mechanism of α2β1 integrin interaction with human echovirus 1

Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of α2β1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a ‘closed’ than an activated ‘open’ form of the α2I domain. Furthermore, a mutation E336A in the α2 subunit, which inactivated α2β1 as a collagen receptor, enhanced α2β1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of α2β1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336‐related conformational changes in α2β1. Furthermore, the mutation E336A did neither prevent EV1 induced and α2β1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on α2β1 clustering, but do not require the conformational regulation of the receptor.

Citrullination of collagen II affects integrin-mediated cell adhesion in a receptor-specific manner

Citrullinated collagen II (CII) is a well‐known autoantigen in rheumatoid arthritis (RA). However, the direct effects of CII citrullination on cell behavior have not been described. To study whether citrullination of CII could affect cellular functions, we measured the adhesion of 3 different cell types (human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal stem cells) with impedance‐based technology. The binding of different collagen receptor integrins to citrullinated collagen was studied by CHO cell lines, each overexpressing 1 of the 4 human collagen receptors on the cell surface, and with solid‐phase binding assays, using the recombinant human integrin α1I, α2I, α10I, and α11I domains. Collagen citrullination decreased the adhesion of synovial fibroblasts ~50% (P<0.05) and mesenchymal stem cells ~40% (P<0.05) by specifically decreasing the binding of integrins α10β1 and α11β1 to arginine‐containing motifs, such as GFOGER. In contrast, citrullination had only a minor effect on the function of α1β1 and α2β1 integrins, which have been reported to play a critical role in regulating leukocyte function. Molecular modeling was used to explain the detected functional differences at the structural level. Given that the integrins regulate cell metabolism, proliferation, and migration, we suggest that collagen citrullination modifies the pathogenesis of RA. Here, CII citrullination was shown to decrease the survival of mesenchymal stem cells.—Sipilä, K., Haag, S., Denessiouk, K., Käpylä, J., Peters, E. C., Denesyuk, A., Hansen, U., Konttinen, Y., Johnson, M. S., Holmdahl, R., Heino, J. Citrullination of collagen II affects integrin‐mediated cell adhesion in a receptor‐specific manner. FASEB J. 28, 3758–3768 (2014). www.fasebj.org

Novel α2β1 Integrin Inhibitors Reveal That Integrin Binding to Collagen under Shear Stress Conditions Does Not Require Receptor Preactivation

Background: Integrin α2β1 is a platelet collagen receptor. Results: Novel sulfonamide derivatives are conformation-selective inhibitors of α2β1, especially when tested under shear stress conditions. Only inhibitors that block non-activated integrins inhibit platelet binding to collagen. Conclusion: Non-activated α2β1 integrin plays an important role in platelet binding to collagen. Significance: We propose an alternative model for α2β1 activation during thrombosis. The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm2). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm2) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.

Histidine-rich glycoprotein blocks collagen-binding integrins and adhesion of endothelial cells through low-affinity interaction with α2 integrin

The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRGs effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRGs inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2β1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.

Extracellular citrullination inhibits the function of matrix associated TGF-β.

In inflammatory arthritis peptidyl arginine deiminase (PAD) enzymes can citrullinate arginine residues in extracellular matrix (ECM) proteins, such as collagens and fibronectin. This may lead to the generation of anti-citrullinated protein antibodies, important diagnostic markers in rheumatoid arthritis. In addition, the citrullination may directly affect protein function. Based on structural analysis, we found that most ECM-associated growth factors (GFs) have arginine residues in their receptor recognition sites. Thus, they are potential functional targets of extracellular citrullination. To examine this further, we focused on the citrullination of transforming growth factor-βs (TGF-β), well-known ECM-associated GFs. PAD-treatment of CHO-LTBP1 cell derived matrix, rich with TGF-β, decreased the level of TGF-β activity as detected by HaCaT and MLEC-PAI-1/Lu reporter cells. Additional experiments indicated that PAD-treatment inhibits the integrin-mediated TGF-β activation since PAD-treatment decreased the binding of integrin αVβ6 ectodomain as well as integrin-mediated spreading of MG-63 and HaCaT cells to β1-latency associated peptide (TGF-β1 LAP). The citrullination of the RGD site, an important integrin recognition motif, was confirmed by mass spectrometry. Furthermore, the citrullination of active TGF-β1 inhibited its binding to recombinant TGF-β receptor II, and prevented its ability to activate TGF-β signaling. Thus, extracellular PAD activity can affect the function of ECM-associated growth factors by different mechanisms. Importantly, the citrullination of both latent and active TGF-β has the potency to regulate the inflammatory process.

18-FDG-PET in a patient cohort suspected for cardiac sarcoidosis: Right ventricular uptake is associated with pathological uptake in mediastinal lymph nodes

Introduction In up to 65% of cardiac sarcoidosis patients, the disease is confined to the heart. Diagnosing isolated cardiac sarcoidosis is challenging due to the low sensitivity of endomyocardial biopsy. If cardiac sarcoidosis is part of biopsy-confirmed systemic sarcoidosis, the diagnosis can be based on cardiac imaging studies. We compared the imaging features of patients with isolated cardiac FDG uptake on positron emission tomography with those who had findings indicative of systemic sarcoidosis. Materials and methods 137 consecutive cardiac FDG-PET/CT studies performed on subjects suspected of having cardiac sarcoidosis were retrospectively analyzed. Results 33 patients had pathological left ventricular FDG uptake, and 12 of these also had pathological right ventricular uptake. 16/33 patients with pathological cardiac uptake had pathological extracardiac uptake. 10/12 patients with both LV- and RV-uptake patterns had extracardiac uptake compared to 6/21 of those with pathological LV uptake without RV uptake. SUVmax values in the myocardium were higher among patients with abnormal extracardiac uptake. The presence of extracardiac uptake was the only imaging-related factor that could predict a biopsy indicative of sarcoidosis. Conclusion Right ventricular involvement seems to be more common in patients who also have findings suggestive of suspected systemic sarcoidosis, compared with patients with PET findings indicative of isolated cardiac disease.

Voluntary liquorice ingestion increases blood pressure via increased volume load, elevated peripheral arterial resistance, and decreased aortic compliance

We investigated the haemodynamic effects of two-week liquorice exposure (glycyrrhizin dose 290–370 mg/day) in 22 healthy volunteers during orthostatic challenge. Haemodynamics were recorded during passive 10-minute head-up tilt using radial pulse wave analysis, whole-body impedance cardiography, and spectral analysis of heart rate variability. Thirty age-matched healthy subjects served as controls. Liquorice ingestion elevated radial systolic (p < 0.001) and diastolic (p = 0.018) blood pressure and systemic vascular resistance (p = 0.037). During orthostatic challenge, heart rate increased less after the liquorice versus control diet (p = 0.003) and low frequency power of heart rate variability decreased within the liquorice group (p = 0.034). Liquorice intake increased central pulse pressure (p < 0.001) and augmentation index (p = 0.002) supine and upright, but in the upright position the elevation of augmentation index was accentuated (p = 0.007). Liquorice diet also increased extracellular fluid volume (p = 0.024) and aortic to popliteal pulse wave velocity (p = 0.027), and aortic characteristic impedance in the upright position (p = 0.002). To conclude, in addition to increased extracellular fluid volume and large arterial stiffness, two weeks of liquorice ingestion elevated systemic vascular resistance and augmentation index. Measurements performed at rest may underestimate the haemodynamic effects of liquorice ingestion, as enhanced central wave reflection and reduced chronotropic response were especially observed in the upright position.

Novel ECG parameters are strongly associated with inflammatory 18F-FDG PET findings in patients with suspected cardiac sarcoidosis

BACKGROUND 18F-Fluorodeoxyglucose (FDG) positron emission tomography (PET) is a feasible method to investigate the inflammatory activity of the myocardium in cardiac sarcoidosis. However, PET is costly and not as widely available as standard electrocardiogram (ECG). Current ECG findings related to cardiac sarcoidosis are highly non-specific. In this study, our aim was to determine whether novel ECG parameters are associated with pathological PET findings in patients with suspected cardiac sarcoidosis. METHODS A total of 133 patients underwent cardiac FDG PET examination in Tampere University Hospital between August 2012 and September 2015. The left ventricular FDG uptake was categorized as either normal or pathological. Additionally, in-depth analyses of resting ECG were performed. Among other parameters, the presence of septal and inferolateral remodelling was assessed. These are novel ECG parameters related to local structural changes in the myocardium. RESULTS In the ECG, septal and inferolateral remodelling, as well as widespread QRS fragmentation were significantly associated with pathological left ventricular FDG uptake even if adjusted for age, sex, body mass index, underlying cardiovascular disease and cardiac medication (p<0.05 for all). When all these ECG parameters were combined in a logistic regression model, only septal remodelling remained independently associated with abnormal left ventricular uptake (p<0.05). CONCLUSIONS Our findings show that novel ECG parameters septal and inferolateral remodelling, as well as diffuse QRS fragmentation, are strongly associated with pathological cardiac findings in FDG PET. Thus, the presence of these ECG findings may warrant the clinician to consider the possibility of cardiac sarcoidosis.

Effect of present versus previous smoking on non-invasive haemodynamics

We examined cardiovascular function in 637 volunteers (19–72 years) without antihypertensive medication in never smokers (n = 365), present smokers (n = 81) and previous smokers (n = 191, median abstinence 10 years). Haemodynamics during passive head-up tilt were recorded using whole-body impedance cardiography and radial pulse wave analysis. Results were adjusted for age, sex, body mass index, LDL cholesterol and alcohol use. Systolic and diastolic blood pressure, heart rate, and pulse wave velocity were not different between the groups. Supine aortic reflection times did not differ, while upright values were shorter in present versus previous smokers (p = 0.04). Heart rate adjusted augmentation index was increased in the supine position in present smokers versus controls (p = 0.045), and in present (p < 0.001) and previous (p = 0.031) smokers versus controls in the upright position. Supine and upright cardiac output was higher (p ≤ 0.016) and systemic vascular resistance lower (p ≤ 0.001) in present versus previous smokers. In spite of the long abstinence, in the upright position previous smokers had lower cardiac output (p = 0.032) and higher systemic vascular resistance (p = 0.014) than never smokers. In the absence of differences in blood pressure and arterial stiffness, present smokers presented with hyperdynamic circulation and enhanced wave reflection compared with previous smokers.

Salbutamol-induced Decrease in Augmentation Index is Related to the Parallel Increase in Heart Rate

The change in augmentation index following salbutamol inhalation has been applied to evaluate endothelial function. We examined the contribution of salbutamol‐induced increase in heart rate to the observed decrease in augmentation index. Haemodynamics were recorded using whole‐body impedance cardiography and continuous pulse wave analysis from tonometric radial blood pressure. All subjects (n = 335, mean age 46, body mass index 26, 48% men) were without medications with cardiovascular influences. The effects of salbutamol inhalation (0.4 mg) versus the endothelium‐independent agent nitroglycerin resoriblet (0.25 mg) were examined during passive head‐up tilt, as the haemodynamic influences of these compounds depend on body position. Salbutamol decreased augmentation index by ~3‐4% units in supine and upright positions. Although salbutamol moderately increased cardiac index (+4.5%) and decreased systemic vascular resistance (−8.5%), the significant haemodynamic explanatory factors for decreased augmentation index in multivariate analysis were increased supine heart rate, and increased upright heart rate and decreased ejection duration (p < 0.001 for all, r2 = 0.36–0.37). Sublingual nitroglycerin decreased supine and upright augmentation index by ~15% units and ~23% units, respectively. The haemodynamic explanatory factors for these changes in multivariate analysis were increased heart rate, reduced ejection duration and reduced systemic vascular resistance (p ≤ 0.021 for all, r2 = 0.22–0.34). In conclusion, the lowering influence of salbutamol on augmentation index may be largely explained by increased heart rate, suggesting that this effect may not predominantly reflect endothelial function.