Stefan Chlopicki

Jagiellonian University | UJ · Jagiellonian Centre for Experimental Therapeutics (JCET)

Adipose triglyceride lipase (ATGL) is involved in lipolysis and displays detrimental pathophysiological role in cardio-metabolic diseases. However, the organo-protective effects of ATGL-induced lipolysis were also suggested. The aim of this work was to characterize the function of lipid droplets (LDs) and ATGL-induced lipolysis in the regulation of...

In this study, we report a series of newly synthesised sulphonamides of aziridine-2-carboxylic acid (Az-COOH) ester and amide analogues as potent protein disulphide isomerase (PDI, EC 5.3.4.1) inhibitors. The inhibitory activity on PDI was determined against recombinant human PDIA1 and PDIA3 proteins using an insulin reduction assay. These compound...

Professional athletes are often exposed to high training loads that may lead to overfatigue, overreaching and overtraining that might have a detrimental effects on vascular health. We determined the effects of high training stress on endothelial function assessed by the flow-mediated dilation (FMD) and markers of glycocalyx shedding. Vascular exami...

Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI2) generation in the vascular tissue of isolated...

Age represents a major risk factor in heart failure (HF). However, the mechanisms linking ageing and HF are not clear. We aimed to identify the functional, morphological and transcriptomic changes that could be attributed to cardiac ageing in a model of slowly progressing HF in Tgαq*44 mice in reference to the cardiac ageing process in FVB mice. In...

Immuno-thrombosis of COVID-19 results in the activation of platelets and coagulopathy. Antiplatelet therapy has been widely used in COVID-19 patients to prevent thrombotic events. However, recent analysis of clinical trials does not support the major effects of antiplatelet therapy on mortality in hospitalized COVID-19 patients, despite the indispu...

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary e...

Accumulation of lipid-laden foam cells in the arterial wall plays a central role in atherosclerotic lesion development, plaque progression, and late-stage complications of atherosclerosis. However, there are still fundamental gaps in our knowledge of the underlying mechanisms leading to foam cell formation in atherosclerotic arteries. Here, we inve...

Endothelial cells line the lumen of all vessels in the body and maintain vascular homeostasis. In particular, endothelial cell regeneration in response to insult sustain functional endothelial layer. EdU (5-ethynyl-2′-deoxyuridine) is an alkyne-tagged proliferation probe that incorporates into newly synthesized DNA and is used for fluorescence imag...

Background Protein disulphide isomerases (PDIs) play an important role in cancer progression. However, the relative contribution of the various isoforms of PDI in tumorigenesis is not clear. Methods The content of PDI isoforms in 22 cancer cells lines was investigated using LC–MS/MS-based proteomic analysis. The effects of PDIA1, PDIA3 and PDIA17...

Einleitung Fluglärm ist mittlerweile ein nachgewiesener kardiovaskulärer Risikofaktor. Entscheidend hierbei ist die Fluglärm-induzierte Entzündungs- reaktion und der oxidative Stress im Gefäßsystem mit begleitender Endothel- zelldysfunktion. Ziel ZieldervorliegendenStudiewareszuklären,obAdpatationoderToler- anzmechanismen bei Mäusen wirksam werden....

Antiplatelet therapy represents a major pharmacological strategy aimed at reducing the risk of atherothrombosis‐associated cardiovascular events. In this chapter, the authors review the evidence supporting the importance of heme oxygenase (HO)‐derived carbon monoxide (CO) in the regulation of thromboresistance in vivo , in particular exemplified by...

The contribution of the shear stress-sensitive epithelial Na ⁺ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to lami...

Fenestrations in liver sinusoidal endothelial cells (LSEC) are transcellular nanopores of 50–350 nm diameter that facilitate bidirectional transport of solutes and macromolecules between the bloodstream and the parenchyma of the liver. Liver diseases, ageing, and various substances such as nicotine or ethanol can negatively influence LSECs fenestra...

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metast...

Transportation noise is recognized as an important cardiovascular risk factor. Key mechanisms are noise-triggered vascular inflammation and oxidative stress with subsequent endothelial dysfunction. Here, we test for adaptation or tolerance mechanisms in mice in response to chronic noise exposure. C57BL/6J mice were exposed to aircraft noise for 0,...

Acyl derivatives of aziridine-2-carboxylic acid have been synthesized and tested as PDIA1 inhibitors. Calculations of charge value and distribution in aziridine ring system and some alkylating agents were performed. For the first time was found that acyl derivatives of aziridine-2-carboxylic acid are weak to moderately active PDIA1 inhibitors.

Cationic amphiphilic drugs (CADs) are known from lysosomotropism, drug-induced phospholipidosis (DIPL), activation of autophagy, and decreased cell viability, but the relationship between these events is not clear and little is known about DIPL in the endothelium. In this work, the effects of fluoxetine, amiodarone, clozapine, and risperidone on hu...

SIRT1, a member of the sirtuin family of longevity regulators, possesses potent activities preventing vascular ageing. The expression and function of SIRT1 in endothelial cells are downregulated with age, in turn causing early vascular ageing and predisposing various vascular abnormalities. Overexpression of SIRT1 in the vascular endothelium preven...

The contribution of the shear-stress sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to lamin...

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR-/-) leads to improvement of exercise capacity. This study aimed to inves...

Background It was previously reported that red blood cells (RBCs) regulate blood flow via RBC-derived ATP [1]. Nevertheless, to the best of our knowledge, no study was performed to characterize possible alterations in RBC ATP levels in ApoE/LDLR−/− mice [2] which constitute a reliable model of human atherosclerosis, displaying distinct erythropathy...

Aims: Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterise the relat...

Glycosaminoglycans (GAGs) constitute the main building blocks of the endothelial glycocalyx (GLX), and disruption of GLX initiates and promotes endothelial dysfunction. Here, we aimed to develop a novel, specific and accurate LC-SRM/MS-based method for glycosaminoglycans (GAGs) profiling. The method involved butanolysis derivatization to facilitate...

Endothelial dysfunction is one of the hallmarks of vascular abnormalities in metabolic diseases and has been repeatedly demonstrated in coronary and peripheral circulation in mice fed high-fat diet (HFD), particularly after long-term HFD. However, the temporal relationship between development of coronary microvascular endothelial dysfunction and de...

A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into str...

Background Platelet-derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. Aims The aim of this study was to characterize the role of PDIA1 in human platelets. Methods Proteomic analysis of PDI isoforms in platelets was performed using LC–MS/MS, and...

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides an indication that NNMT may be an interesting therapeutic target for a variety of disease...

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways...

The goal of this study was to assess the alterations in left ventricle (LV) blood flow velocity patterns in relation to progressive impairment of cardiac function in the course of heart failure (HF) in a unique murine model of chronic HF in Tgαq*44 mice. Doppler- and MRI-based assessments of blood velocity and cardiac performance, respectively, wer...

Vitamins K exert a range of activities that extend far beyond coagulation and include anti-inflammatory effects, but the mechanisms involved in anti-inflammatory action remain unclear. In the present study, we showed that various forms of exogenous vitamins—K1, K3, K2 (MK-4, MK-5, MK-6 and MK-7)—regulated a wide scope of inflammatory pathways in mu...

Hyperglycemia linked to diabetes results in endothelial dysfunction. In the present work, we comprehensively characterized effects of short-term hyperglycemia induced by administration of an insulin receptor antagonist, the S961 peptide, on endothelium and perivascular adipose tissue (PVAT) in mice. Endothelial function of the thoracic and abdomina...

Angiotensin-converting enzyme inhibition (ACE-I) and physical activity favorably modulate the ACE/ACE-2 balance. However, it is not clear whether physical activity and ACE-I could synergistically modulate ACE/ACE-2 balance in the course of heart failure (HF). Here, we studied the effects of combined spontaneous physical activity and ACE-I–based tre...

Endothelial inflammation is the hallmark of vascular pathology often proceeding with cardiovascular diseases. Here, we adopted a multiparameter approach combining various imaging techniques at the nano- and microscale (Raman, AFM and fluorescence) to investigate endothelial inflammation in response to lipopolysaccharides (LPS) in vitro in human mic...

Arterial hypertension is one of the major health risk factors leading to coronary artery disease, stroke or peripheral artery disease. Dietary uptake of inorganic nitrite (NO2⁻) and nitrate (NO3⁻) via vegetables leads to enhanced vascular NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying v...

Nicotinamide adenine dinucleotide (NAD+) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto‐enzymes in controlling adenine nucleotide–dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD+ and mononucleotide nicotinamide...

Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and te...

Background: Critical limb ischemia (CLI) is the most advanced stage of peripheral arterial disease. CLI patients, compared to non-CLI, achieve worse treatment outcomes and generate higher costs. Aims: The aim of the study was to compare endothelial function and clinical outcomes in CLI and non-CLI patients after percutaneous transluminal angiopl...

Raman spectroscopy is successfully becoming an analytical tool used to characterize alterations in the biochemical composition of cells. In this work, we identify the features of Raman spectra of murine primary endothelial cells (EC) isolated from lungs, heart, liver, brain, kidney and aorta of normal mice, as well as from heart, lung and liver in...

Most, if not all, of the hitherto tested substances exert more or less pronounced pro-survival effects when applied before or immediately after the exposure to high doses of ionizing radiation. In the present study we demonstrate for the first time that 1-methyl nicotinamide (MNA), a derivative of vitamin B 3 , significantly (1.6 to 1.9 times) prol...

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is need...

In this study we characterize the impact of aging on the spontaneous running performance of the Tgαq*44 mice (transgenic murine model of chronic heart failure) as compared to the wild-type FVB mice. In 166 mice we have recorded the following parameters of their physical activities in the running wheels: the total distance covered during the experim...

Carbon monoxide (CO)—gaseous or released by CO-RMs—both possess antiplatelet properties; however, it remains uncertain whether the mechanisms involved are the same. Here, we characterise the involvement of soluble guanylate cyclase (sGC) in the effects of CO—delivered by gaseous CO–saturated buffer (COG) and generated by CORM-A1—on platelet aggrega...

Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome–lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1)...

Background: Vascular drug delivery becomes a promising direction in the development of novel therapeutic strategies in the treatment of cardiovascular pathologies, such as hypertension. However, targeted delivery of hydrophobic substances, with poor bioavailability, remains a challenge. Here, we described the hypotensive effects of a low dose of c...

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by progressive destruction of the intrahepatic bile ducts. The immunopathology of PBC involves excessive inflammation; therefore, negative regulators of inflammatory response, such as Monocyte Chemoattractant Protein-1-Induced Protein-1 (MCPIP1) may play important roles in the...

Background Dyslipidaemia is a major risk factor for atherosclerosis and cardiovascular diseases. The molecular mechanisms that translate dyslipidaemia into atherogenesis and reliable markers of its progression are yet to be fully elucidated. To address this issue, we conducted a comprehensive metabolomic and proteomic analysis in an experimental mo...

In this work, the effect of an early oxidative stress on human endothelial cells induced by menadione was studied using a combined methodology of label-free Raman imaging and fluorescence staining. Menadione-induced ROS-dependent endothelial inflammation in human aorta endothelial cells (HAEC) was studied with focus on changes in cytochrome, protei...

Perivascular adipose tissue (PVAT) regulates vascular function and represents a novel therapeutic target in vascular diseases. In this work, a new approach based on fiber-optic Raman spectroscopy and spectral modelling was used to characterize the chemical content of the PVAT of the internal mammary artery (IMA) of patients with advanced coronary a...

Objective Dyslipidaemia is a major risk factor for myocardial infarction that is known to correlate with atherosclerosis in the coronary arteries. We sought to clarify whether metabolic alterations induced by dyslipidaemia in cardiomyocytes collectively constitute an alternative pathway that escalates myocardial injury. Methods Dyslipidaemic apoli...

BACKGROUND: Long-term feeding with a high-fat diet (HFD) induces endothelial dysfunction in mice, but early HFD-induced effects on endothelium have not been well characterized. METHODS AND RESULTS: Using an magnetic resonance imaging-based methodology that allows characterization of endothelial function in vivo, we demonstrated that short-term (2 w...

Histamine is a pleiotropic biogenic amine, having affinity towards four distinct histamine receptors. The existing pharmacological studies suggest the usefulness of histamine H4 receptor ligands in the treatment of many inflammatory and immunomodulatory diseases, including allergic rhinitis, asthma, atopic dermatitis, colitis or pruritus. Up to dat...

Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in prol...

In this work we applied a multimodal approach to define the age- and atherosclerosis-related biochemical and functional alterations in red blood cells (RBCs) in ApoE/LDLR-/- mice. Our results revealed that age-related changes in RBCs, such as decreases in RBC deformability and mean height, were more pronounced in ApoE/LDLR-/- mice than in age-match...

Background & Aims Primary biliary cholangitis (PBC) is an autoimmune disease characterized by progressive destruction of the intrahepatic bile ducts. The immunopathology of PBC involves excessive inflammation; therefore, negative regulators of inflammatory response, such as Monocyte Chemoattractant Protein-1-Induced Protein-1 (MCPIP1, alias Regnase...

Objectives Carbon monoxide (CO) produced by haem oxygenases or released by CO releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO–induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results To...

Based on the evidence supporting platelet-dependent mechanisms protecting endothelial barrier integrity in inflammation, it is tempting to speculate that increased microcirculation permeability associated with thrombocytopenia or prolonged use of antiplatelet agents and preexistising endothelial dysfunction of cardiovascular diseases may contribute...