Graphene Coating Obtained in a Cold-Wall CVD Process on the Co-Cr Alloy (L-605) for Medical Applications.

Graphene coating on the cobalt-chromium alloy was optimized and successfully carried out by a cold-wall chemical vapor deposition (CW-CVD) method. A uniform layer of graphene for a large area of the Co-Cr alloy (discs of 10 mm diameter) was confirmed by Raman mapping coated area and analyzing specific G and 2D bands; in particular, the intensity ratio and the number of layers were calculated. The effect of the CW-CVD process on the microstructure and the morphology of the Co-Cr surface was investigated by scanning X-ray photoelectron microscope (SPEM), atomic force microscopy (AFM), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). Nanoindentation and scratch tests were performed to determine mechanical properties of Co-Cr disks. The results of microbiological tests indicate that the studied Co-Cr alloys covered with a graphene layer did not show a pro-coagulant effect. The obtained results confirm the possibility of using the developed coating method in medical applications, in particular in the field of cardiovascular diseases.

Graphene Oxide Carboxymethylcellulose Nanocomposite for Dressing Materials.

Sore, infected wounds are a major clinical issue, and there is thus an urgent need for novel biomaterials as multifunctional constituents for dressings. A set of biocomposites was prepared by solvent casting using different concentrations of carboxymethylcellulose (CMC) and exfoliated graphene oxide (Exf-GO) as a filler. Exf-GO was first obtained by the strong oxidation and exfoliation of graphite. The structural, morphological and mechanical properties of the composites (CMCx/Exf-GO) were evaluated, and the obtained composites were homogenous, transparent and brownish in color. The results confirmed that Exf-GO may be homogeneously dispersed in CMC. It was found that the composite has an inhibitory activity against the Gram-positive Staphylococcus aureus, but not against Gram-negative Pseudomonas aeruginosa. At the same time, it does not exhibit any cytotoxic effect on normal fibroblasts.

Functionalization with a VEGFR2-binding antibody fragment leads to enhanced endothelialization of a cardiovascular stent in vitro and in vivo.

Rapid endothelialization of cardiovascular stents is critical to prevent major clinical complications such as restenosis. Reconstruction of the native endothelium on the stent surface can be achieved by the capture of endothelial progenitor cells (EPCs) or neighboring endothelial cells (ECs) in vivo. In this study, stainless steel cardiovascular stents were functionalized with recombinant scFv antibody fragments specific for vascular endothelial growth factor receptor-2 (VEGFR2) that is expressed on EPCs and ECs. Anti-VEGFR2 scFvs were expressed in glycosylated form in Escherichia coli and covalently attached to amine-functionalized, titania-coated steel disks and stents. ScFv-coated surfaces exhibited no detectable cytotoxicity to human ECs or erythrocytes in vitro and bound 15 times more VEGFR2-positive human umbilical vein ECs than controls after as little as 3 min. Porcine coronary arteries were successfully stented with scFv-coated stents with no adverse clinical events after 30 days. Endovascular imaging and histology revealed coverage of the anti-VEGFR2 scFv-coated stent with a cell layer after 5 days and the presence of a neointima layer with a minimum thickness of 80 µm after 30 days. Biofunctionalization of cardiovascular stents with endothelial cell-capturing antibody fragments in this manner offers promise in accelerating stent endothelialization in vivo. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:213-224, 2020.

Photoactive Pore Matrix for In Situ Delivery of a Photosensitizer in Vascular Smooth Muscle Cells Selective PDT.

In this study we present the porous silica-based material that can be used for in situ drug delivery, offering effective supply of active compounds regardless its water solubility. To demonstrate usability of this new material, three silica-based materials with different pore size distribution as a matrix for doping with Photolon (Ph) and Protoporphyrin IX (PPIX) photosensitizers, were prepared. These matrices can be used for coating cardiovascular stents used for treatment of the coronary artery disease and enable intravascular photodynamic therapy (PDT), which can modulate the vascular response to injury caused by stent implantation-procedure that should be thought as an alternative for drug eluting stent. The FTIR spectroscopic analysis confirmed that all studied matrices have been successfully functionalized with the target photosensitizers. Atomic force microscopy revealed that resulting photoactive matrices were very smooth, which can limit the implantation damage and reduce the risk of restenosis. No viability loss of human peripheral blood lymphocytes and no erythrocyte hemolysis upon prolonged incubations on matrices indicated good biocompatibility of designed materials. The suitability of photoactive surfaces for PDT was tested in two cell lines relevant to stent implantation: vascular endothelial cells (HUVECs) and vascular smooth muscle cells (VSMC). It was demonstrated that 2 h incubation on the silica matrices was sufficient for uptake of the encapsulated photosensitizers. Moreover, the amount of the absorbed photosensitizer was sufficient for induction of a phototoxic reaction as shown by a rise of the reactive oxygen species in photosensitized VSMC. On the other hand, limited reactive oxygen species (ROS) induction in HUVECs in our experimental set up suggests that the proposed method of PDT may be less harmful for the endothelial cells and may decrease a risk of the restenosis. Presented data clearly demonstrate that porous silica-based matrices are capable of in situ delivery of photosensitizer for PDT of VSMC.

Photoactive Liposomal Formulation of PVP-Conjugated Chlorin e6 for Photodynamic Reduction of Atherosclerotic Plaque.

BACKGROUND: Liposomes serve as delivery systems for biologically active compounds. Existing technologies inefficiently encapsulate large hydrophilic macromolecules, such as PVP-conjugated chlorin e6 (Photolon). This photoactive drug has been widely tested for therapeutic applications, including photodynamic reduction of atherosclerotic plaque. METHODS: A novel formulation of Photolon was produced using "gel hydration technology". Its pharmacokinetics was tested in Sus scrofa f. domestica. Its cellular uptake, cytotoxicity, and ability to induce a phototoxic reaction were demonstrated in J774A.1, RAW264.7 macrophages, and vascular smooth muscle (T/G HA-VSMC) as well as in vascular endothelial (HUVEC) cells. RESULTS: Developed liposomes had an average diameter of 124.7 ± 0.6 nm (polydispersity index (PDI) = 0.055) and contained >80% of Photolon). The half-life of formulation in S. scrofa was 20 min with area under the curve (AUC) equal to 14.7. The formulation was noncytotoxic in vitro and was rapidly (10 min) and efficiently accumulated by macrophages, but not T/G HA-VSMC or HUVEC. The accumulated quantity of photosensitizer was sufficient for induction of phototoxicity in J774A.1, but not in T/G HA-VSMC. CONCLUSIONS: Due to the excellent physical and pharmacokinetic properties and selectivity for macrophages, the novel liposomal formulation of Photolon is a promising therapeutic candidate for use in arteriosclerosis treatment when targeting macrophages but not accompanying vascular tissue is critical for effective and safe therapy.

Functionalized CD133 antibody coated stent surface simultaneously promotes EPCs adhesion and inhibits smooth muscle cell proliferation-A novel approach to prevent in-stent restenosis.

We report a multistep strategy of biochemical surface modifications that resulted in the synthesis of new, effective and biocompatible intravascular implants coating with immobilized anti-CD133 antibodies, that proved to be the most effective in endothelial progenitor cells capture and reduced smooth muscle cells growth. Biomolecules were immobilized on differently functionalized surfaces. The distribution, nanostructural characteristics and intramolecular interactions of anti-CD133 molecules as well as their ability to bind EPCs was evaluated. We also tempted to build a molecular model of the CD133 protein to study antigen-antibody interactions. CD133 protein is expressed in endothelial progenitor cells (EPCs). Absence of preferential interaction site on CD133, but rather a presence of a small binding area, may be the specificity of reconnaissance sequence, thus importantly increasing the probability of CD133 protein binding. After all, regarding our molecular model, we are convinced that specific, and large enough interactions between anti-CD133 coating stent surface and CD133 present on EPCs will reduce risk of restenosis by favoring the endothelial growth. Additionally, the safety study of the vivo performance of modified titania based surface was performed using small animal models. No allergological or toxical local or systemic adverse effects of the developed coatings were noted.

Biocompatible Carbon-Based Coating as Potential Endovascular Material for Stent Surface.

Stainless steel 316L is a material commonly used in cardiovascular medicine. Despite the various methods applied in stent production, the rates of in-stent restenosis and thrombosis remain high. In this study graphene was used to coat the surface of 316L substrate for enhanced bio- and hemocompatibility of the substrate. The presence of graphene layers applied to the substrate was investigated using cutting-edge imaging technology: energy-filtered low-voltage FE-SEM approach, scanning electron microscopy (SEM), Raman spectroscopy, and atomic force microscopy (AFM). The potential of G-316L surface to influence endothelial cells phenotype and endothelial-to-mesenchymal transition (EndoMT) has been determined. Our results show that the bio- and hemocompatible properties of graphene coatings along with known radial force of 316L make G-316L a promising candidate for intracoronary implants.

Low frequency electromagnetic field decreases ischemia-reperfusion injury of human cardiomyocytes and supports their metabolic function.

Electromagnetic field at extremely low frequencies plays a significant role in the physiological function of human tissues and systems. It is shown that electromagnetic field inhibits production of reactive oxygen species which are involved in heart injury triggered by oxidative stress. We hypothesize that low frequency electromagnetic field protects function of cardiac cells from ischemia-reperfusion injury. Human cardiac myocytes, endothelial cells, and cardiac fibroblast underwent ischemia-reperfusion conditions in the presence or in the absence of low frequency electromagnetic field. LDH and MMP-2 activities (as markers of cell injury), and cell metabolic activity (by fluorescein diacetate staining) were measured to determine the protective role of low frequency electromagnetic field. Our data showed that short courses of low frequency electromagnetic field protect cardiac cells from cellular damage and preserve their metabolic activity during ischemia-reperfusion. This study demonstrates the possibility to use of low frequency electromagnetic field as strategy for the prevention or therapy of ischemia-reperfusion injury. Impact statement In our study, we showed that LF-EMF may be protective for heart during ischemia-reperfusion (I/R). Following is the short description of the main findings: (a) the response to the I/R injury was different for endothelial cells, fibroblasts, and cardiomyocytes; (b) I/R decreases MMP-2 activity in cardiac myocytes and fibroblasts;

Multimodality imaging of intermediate lesions: Data from fractional flow reserve, optical coherence tomography, near-infrared spectroscopy-intravascular ultrasound.

BACKGROUND: Fractional flow reserve (FFR) assesses a functional impact of the atheroma on the myocardial ischemia, but it does not take into account the morphology of the lesion. Previous optical coherence tomography (OCT), intravascular ultrasound (IVUS) and near-infrared spectroscopy (NIRS) studies presented their potential to detect vulnerable plaques, which is not possible by FFR assessment. With the following study, the intermediate lesions were assessed by FFR, OCT and combined NIRS-IVUS imaging to identify plaque vulnerability. METHODS: Thirteen intermediate lesions were analyzed simultaneously by FFR, OCT and combined NIRS-IVUS imaging. RESULTS: Two lesions were found to have FFR ≤ 0.80 (0.65 and 0.76). The other 11 lesions had FFR > 0.80 with a mean FFR 0.88 ± 0.049. Two lesions with FFR ≤ 0.80 had plaque burden (PB) > 70% and minimal lumen area (MLA) < 4 mm2, but neither of these 2 lesions were identified as OCT de-fined thin fibrous cap atheroma (TCFA), or NIRS-IVUS possible TCFA. Among the other 11 lesions with FFR > 0.80, 8 were identified as OCT-defined TCFA, 4 had PB > 70%, 6 had MLA < 4 mm2, 2 had both PB > 70% and MLA < 4 mm2, 3 lesions were identified as NIRS-IVUS possible TCFA, and 4 lesions had lipid core burden index > 400. CONCLUSIONS: The FFR-negative lesions pose traits of vulnerability as assessed simultaneously by IVUS, OCT and NIRS imaging.

L-NAME improves doxycycline and ML-7 cardioprotection from oxidative stress.

Matrix metalloproteinase-2 (MMP-2) mediated degradation of myosin light chain 1 (MLC1) and troponin I (TnI) contributes to myocardial ischemia/reperfusion (I/R) injury. Modifications of MLC1 triggered by oxidative stress are mediated by myosin light chain kinase (MLCK), nitric oxide synthase (NOS), and MMP-2. Previous studies have shown that inhibiting both MLCK and MMP-2 protects against I/R injury. Here, we hypothesized that the addition of NOS inhibitor (L-NAME) at subprotective concentration to the mixture of subprotective concentrations of ML-7 and doxycycline (Doxy), will increase a synergistic cardioprotection of Doxy and ML-7 during I/R. Isolated rat hearts were subjected to global ischemia without or with administration of the mixture of inhibitors. Markers of I/R injury were measured in hearts and coronary effluents. Addition of L-NAME to the mixture of Doxy and ML-7 led to full recovery of heart contractility in comparison to combination of Doxy and ML-7. Improved heart contractility was associated with reduced degradation of TnI and MLC1. The combined administration of NOS, MMP-2 and MLCK inhibitors provides a novel strategy to protect heart from I/R injury.

Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP-2/MLCK-mediated cardiomyocyte protection from hypoxic injury.

Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase-2 (MMP-2). It has been shown that simultaneous inhibition of MMP-2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML-7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co-administration of nitric oxide synthase (NOS) inhibitor (1400W or L-NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia-reoxygenation (H-R)-induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP-2 by Doxy (25-100 µM), MLCK by ML-7 (0.5-5 µM) and NOS by L-NAME (25-100 µM) or 1400W (25-100 µM) protected myocyte contractility after H-R in a concentration-dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H-R at the level of highest single-drug concentration. The combination of subthreshold concentrations of NOS, MMP-2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP-2. The observed protection with addition of L-NAME or 1400W was better than previously reported combination of ML-7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility.

Low Frequency Electromagnetic Field Conditioning Protects against I/R Injury and Contractile Dysfunction in the Isolated Rat Heart.

Low frequency electromagnetic field (LF-EMF) decreases the formation of reactive oxygen species, which are key mediators of ischemia/reperfusion (I/R) injury. Therefore, we hypothesized that the LF-EMF protects contractility of hearts subjected to I/R injury. Isolated rat hearts were subjected to 20 min of global no-flow ischemia, followed by 30 min reperfusion, in the presence or absence of LF-EMF. Coronary flow, heart rate, left ventricular developed pressure (LVDP), and rate pressure product (RPP) were determined for evaluation of heart mechanical function. The activity of cardiac matrix metalloproteinase-2 (MMP-2) and the contents of coronary effluent troponin I (TnI) and interleukin-6 (IL-6) were measured as markers of heart injury. LF-EMF prevented decreased RPP in I/R hearts, while having no effect on coronary flow. In addition, hearts subjected to I/R exhibited significantly increased LVDP when subjected to LF-EMF. Although TnI and IL-6 levels were increased in I/R hearts, their levels returned to baseline aerobic levels in I/R hearts subjected to LF-EMF. The reduced activity of MMP-2 in I/R hearts was reversed in hearts subjected to LF-EMF. The data presented here indicate that acute exposure to LF-EMF protects mechanical function of I/R hearts and reduces I/R injury.

Biventricular response of the heart to endurance exercise training in previously untrained subjects.

BACKGROUND: Functional adaptation of the heart to regular strenuous exercise has not been fully elucidated yet, with different patterns of alterations being reported. We evaluated the effect of endurance exercise training (EET) on left (LV) and right ventricular (RV) mechanics in amateur individuals preparing for triathlon competitions. METHODS: Twenty-one subjects aged 33 ± 6 years underwent conventional and speckle tracking echocardiography at rest before and after a high-intensity (12.3 ± 1.0 h/week) 12-month EET. RESULTS: At follow-up, in addition to the improvement in LV diastolic parameters, a significant decrease in longitudinal (26.0 ± 3.3% vs. 24.3 ± 3.2%, P < 0.04), circumferential (24.3 ±4.3% vs. 20.1 ± 3.8%, P < 0.002), and radial strains (46.8 ± 18.3% vs. 37.8 ± 12.9%, P < 0.03), and rotation (9.7 ± 4.8% vs. 7.1 ± 4.0 deg, P < 0.04) was demonstrated at the apex, whereas the LV base was found to show an increase in rotation (-3.9 ± 2.8% vs. -5.9 ± 1.8 deg, P < 0.01). Overall hemodynamic effectiveness of the LV was preserved, as evidenced by the unchanged ejection fraction, cardiac output, twist, and torsion. RV systolic function as assessed by strain was significantly reduced with EET (28.1 ± 6.7% vs. 23.7 ± 8.6%, P < 0.03). CONCLUSIONS: EET modifies both LV and RV performance at rest in previously untrained subjects. The true nature of these changes (adaptive or maladaptive) is unclear, but the hypothesis of different responses of the LV apex and base, with the reduction in contractility of the former and increase in rotation of the latter, representing a protective mechanism that reduces myocardial stress might be considered.

Inhibition of MMP-2 expression affects metabolic enzyme expression levels: proteomic analysis of rat cardiomyocytes.

In this study we examined the effect of inhibition of MMP-2 expression, using siRNA, on the cardiomyocyte proteome. Isolated cardiomyocytes were transfected with MMP-2 siRNA and incubated for 24h. Control cardiomyocytes from the same heart were transfected with scrambled siRNA following the same protocol. Comparison of control cardiomyocyte proteomes with proteomes from MMP-2 suppressed cardiomyocytes revealed 13 protein spots of interest (9 protein spots increased; 4 decreased). Seven protein spots were identified as mitochondrial enzymes involved in energy production and represent: ATP synthase beta subunit, dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, cytochrome c oxidase subunit 5A, electron transfer flavoprotein subunit beta, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 5 and a fragment of mitochondrial precursor of long-chain specific acyl-CoA dehydrogenase. Furthermore, precursor of heat shock protein 60 and Cu-Zn superoxide dismutase were identified. Two protein spots corresponding to MLC1 were also detected. In addition, ATP synthase activity was measured and was increased by approximately 30%. Together, these results indicate that MMP-2 inhibition represents a novel cardioprotective therapy by promoting alterations in the levels of mitochondrial enzymes for improved energy metabolism and by preventing degradation of contractile proteins needed for normal excitation-contraction coupling. BIOLOGICAL SIGNIFICANCE: During ischemia and reperfusion of cardiomyocytes, abnormality in excitation-contraction coupling and decreased energy metabolism often lead to myocardial infarction, but the cellular mechanisms are not fully elucidated. We show for the first time that intracellular inhibition of MMP-2 in cardiomyocytes increases contractility of aerobically perfused myocytes, which was accompanied by increased expression of contractile proteins (e.g., MLC-1). We also showed that MMP-2 inhibition produced a cardiomyocyte proteome that is consistent with improved mitochondrial energy metabolism (e.g., increased expression and activity of mitochondrial beta ATP synthase). Thus, MMP-2 appears to be involved in homeostatic regulation of protein turnover. Our results are significant since they point to targeting MMP-2 activity as a novel therapeutic option to limit myocardial damage by decreasing proteolytic degradation of mitochondrial metabolic enzymes and myocardial contractile proteins during ischemia. In addition, the development of novel pharmacological agents that selectively targets cardiac MMP-2 represents a novel approach to treat and prevent other heart diseases.

In vitro photodynamic therapy with chlorin e6 leads to apoptosis of human vascular smooth muscle cells.

Percutaneous coronary intervention has become the most common and widely implemented method of heart revascularization. However, the development of restenosis remains the major limitation of this method. Photodynamic therapy (PDT) recently emerged as a new and promising method for the prevention of arterial restenosis. Here the efficacy of chlorin e6 in PDT was investigated in vitro using human vascular smooth muscle cells (TG/HA-VSMCs) as one of the cell types crucial in the development of restenosis. PDT-induced cell death was studied on many levels,including annexin V staining, measurement of the generation reactive oxygen species (ROS) and caspase-3 activity,and assessment of changes in mitochondrial membrane potential and fragmentation of DNA. Photosensitization of TG/HA-VSMCs with a 170 lM of chlorin e6 and subsequent illumination with the light of a 672-nm diode laser(2 J/cm2) resulted in the generation of ROS, a decrease in cell membrane polarization, caspase-3 activation, as well as DNA fragmentation. Interestingly, the latter two apoptotic events could not be observed in photosensitized and illuminated NIH3T3 fibroblasts, suggesting different outcomes of the model of PDT in various types of cells. The results obtained with human VSMCs show that chlorin e6 may be useful in the PDT of aerial restenosis, but its efficacy still needs to be established in an animal model.

Endoluminal phototherapy for prevention of restenosis: preliminary results at 6-month follow-up.

OBJECTIVE: The objective of this observational study was to investigate safety and efficacy of laser phototherapy (LPh) in prevention of restenosis after percutaneous coronary intervention (PCI). BACKGROUND DATA: Laser irradiation is known to cause a limitation of the local inflammatory cascade and a stimulation of proliferation of specific cells. Based on the results of previous experiments proving the beneficial effects of laser light on the activity of vascular and inflammatory cells, we attempted to use these properties to prevent restenosis. METHODS: Laser phototherapy was performed in 41 patients after stent implantation or balloon angioplasty. Illumination power of 100 mW and energy dose equal to 9 J/cm(2) was used. Patients were monitored for major adverse cardiac events (MACE) after 30 days and 6 months. At 6 months, angiography as a control was performed to assess the influence of LPh on restenosis rate. RESULTS: Angiographic follow-up (n = 30) revealed restenosis in 9% and 25% of patients after stent implantation and balloon angioplasty, respectively. The MACE rate was 4.5% and 12.5% in stent and balloon-treated patients, respectively. CONCLUSIONS: Laser phototherapy gives very promising results in restenosis prevention, especially after stent implantation. The treatment method is safe, with a low rate of MACE in follow-up.

[Successful angioplasty of the left anterior descending artery in a woman with Takayasu arteritis and associated coronary disease]. / Choroba Takayasu czynnikiem ryzyka progresji miazdzycy? Skuteczny zabieg angioplastyki wiencowej u pacjentki z oboma schorzeniami.

Takayasu arteritis is a rare, inflammatory disease affecting mainly young women and is more prevalent in Asia and Latin America. Its etiology is obscure. It involves the aorta and its main branches. The typical lesions are represented by obliteration and aneurysmatic dilatation of arteries and ostial stenosis. We report a case of a 51-year-old woman with Takayasu arteritis and coronary disease, complaining of angina pectoris CCS class IV. The patient underwent successful angioplasty of LAD. During 6-month follow-up she remained asymptomatic. Various therapeutic options of revascularization are reviewed.

[Pericarditis as a consequence and first symptom of microcellular lung carcinoma--case report]. / Zapalenie osierdzia jako powiklanie i pierwszy objaw raka drobnokomórkowego oskrzeli.

A case of a patient suffering from Streptococcal suppurative pericarditis with cardiac tamponade and acute renal failure as a first symptom of disseminated lung cancer is presented. The examination of pericardial fluid only confirmed Streptococcal infection as a cause of exudative pericarditis. The neoplasmatic cells were not found in the examined fluid. Next, chest CT and bronchoscopy were performed due to the lack of clinical improvement, recurrence of severe pericardial effusion and enlargement of the upper mediastinum found on chest radiogram. CT and bronchoscopy allowed to establish the final diagnosis - disseminated lung cancer.